Systems for in vivo site-directed mutagenesis using oligonucleotides

ABSTRACT

This disclosure provides several methods to generate nucleic acid mutations in vivo, for instance in such a way that no heterologous sequence is retained after the mutagenesis is complete. The methods employ integrative recombinant oligonucleotides (IROs). Specific examples of the described mutagenesis methods enable site-specific point mutations, deletions, and insertions. Also provided are methods that enable multiple rounds of mutation and random mutagenesis in a localized region. The described methods are applicable to any organism that has a homologous recombination system.

PRIORITY CLAIM

[0001] This application claims the benefit of co-pending U.S.Provisional Patent Application No. 60/308,426, filed Jul. 27, 2001,which is incorporated here in its entirety.

FIELD

[0002] This disclosure relates to methods of in vivo site-directedmutagenesis, particularly mutagenesis mediated by oligonucleotides. Itfurther relates to methods of in vivo site-directed mutagenesis that arestimulated by double-strand breaks induced in vivo and mediated byoligonucleotides.

BACKGROUND OF THE DISCLOSURE

[0003] The combination of genome sequencing along with commonality ofgenetic function across species is providing an opportunity tocharacterize the genes of higher organisms in two ways: by analyzingsite-specific changes in homologous genes in model systems and throughanalysis of higher eukaryotic genes cloned within model organisms. Theyeast Saccharomyces cerevisiae has proven ideal for many cross-speciesstudies. The growing body of knowledge and techniques that have made itthe best-characterized eukaryotic genome (Dujon, Trends Genet. 12,263-270, 1996; Hieter et al., Nat.Genet 13, 253-255, 1996; Resnick andCox, Mutat. Res. 451, 1-11, 2000; Shashikant et al., Gene 223, 9-20,1998) also allow the experimental manipulation of large heterologousgenomic DNA fragments cloned into yeast artificial chromosomes (YACs)(Anand, Trends Biotechnol. 10, 35-40, 1992, Larionov et al, Proc. Natl.Acad. Sci. USA 94, 7384-7387, 1997; Brown et al., Trends Biotechnol. 18,218-23, 2000).

[0004] While it is possible in yeast to modify natural chromosomes orYACs without leaving behind any heterologous sequence, present systemshave limited flexibility and are laborious. For example, null mutationsare usually made in yeast by gene replacement, such that a marker genereplaces the sequence that is deleted (Wach et al., Yeast 10, 1793-1808,1994). Marker recycling procedures, based on homologous or site-specificrecombination or religation of DNA ends, also leave heterologousmaterial at the deleted locus, such as hisG (Alani et al, Genetics 116,541-545, 1987), FRT (Storici et al., Yeast 15, 271-283, 1999), loxP(Delneri et al., Gene 252, 127-135, 2000), or l-Scel (Fairhead et al.,Yeast 12, 1439-1457, 1996) sequences. To accomplish sequencemodification such that no heterologous material is retained requiressubcloning and in vitro mutagenesis (Scherer and Davis, Proc. Natl. AcadSci. USA 76, 4949-4955, 1979; Barton et al., Nucleic Acids Res. 18,7349-7355, 1990). PCR-based procedures that do not involve cloning areinefficient or require multistep reactions which increase the risk ofadditional mutations (Langle-Rouault and Jacobs, Nucleic Acids Res. 23,3079-3081, 1995; Erdeniz et al., Genome Res. 7, 1174-1183, 1997). Analternative approach has been demonstrated in yeast for the CYCl genethat relies on transformation with an oligonucleotide (Moerschell etal., Proc. Natl. Acad. Sci. USA 85, 524-528, 1988), but the method isrestricted to the generation of mutants with a selectable phenotype andappears to be target dependent (Kmiec, “Targeted generepair”[editorial], Gene Ther. 6, 1-3, 1999). Oligonucleotides, whencombined with gap repair, can also be used to modify plasmids in yeast(Duno et al., Nucleic Acids Res. 27:el, 1999); however, this approach islimited by restriction site availability.

SUMMARY OF THE DISCLOSURE

[0005] This disclosure provides simple and effective methods that usepurified or unpurified oligonucleotides to generate specific mutationsin vivo, for instance, in yeast chromosomal DNAs, in such a way that noheterologous sequence is retained after the mutagenesis is complete.Specific methods provide for site-specific mutagenesis, including singleor multiple point mutations, short deletions, or short insertions. Alsoprovided are methods for more extensive deletions of precise nucleicacid sequences. A variety of individual and complementaryoligonucleotides have been examined, and parameters that influencesite-directed mutagenesis (e.g., oligonucleotide length) are described.Also provided are methods that enable multiple rounds of mutation andrandom mutagenesis in a localized region. The mutagenesis approaches areapplicable to all organisms where homologous recombination is or can becarried out.

[0006] The two-step, cloning-free methods generate mutated products invivo having only the desired mutation, such as single or multiple basechanges, an insertion, or a small or a large deletion. Delitto perfetto(idiom used to represent “perfect deletion”) mutagenesis is extremelyversatile. It enables multiple rounds of specific or random changeswithin a specified window of 200 bp or more. Since yeast is commonlyused for random and selective cloning of genomic DNA from highereukaryotes such as YACs, this strategy also provides an efficient way tocreate precise changes within mammalian or other heterologous DNAsequences.

[0007] This disclosure further provides tools, including specificcassettes and classes of cassettes, that expand the applicability of thedelitto perfetto system to virtually all yeast strains, independent fromtheir genetic background. Specific embodiments include creation of largechromosomal deletions, up to 16 kb deletions having high accuracy. A setof four alternative specific CORE-cassettes are disclosed, which can beused to make the system applicable for all haploid yeast strains,including wild type non-auxotrophic strains.

[0008] In addition to the common heterologous URA3Kl, G418, andHygromycin resistance genes, a novel counter-selectable marker isdeveloped and disclosed, based on the human p53 allele V122A (Inga etal., Oncogene 20: 3409-3419, 2001). The delitto perfetto mutagenesissystem can be used in diploid as well as haploid strains. The alteredtransactivation specificity of this allele prevents growth whenoverexpressed, without affecting genome stability.

[0009] In addition, this disclosure presents modifications of thedelitto perfetto system that provides dramatically improvedrecombination efficiency, in some embodiments over 1000-fold increase inefficiency. These embodiments, using methods termed generally delittoperfetto-DSB, exploit the use of induced site-specific DSBs in thegenome to increase oligonucleotide targeted mutagenesis. Specificexamples of DSB-enhanced delitto perfetto mutagenesis do not require,and do not include, the use of a counterselectable marker.

[0010] The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 is a schematic drawing illustrating two embodiments of thedelitto perfetto system using integrative recombinant oligonucleotides(IROs); one embodiment illustrates the deletion of a sequence, and theother illustrates the creation of a specific point mutation.

[0012] In Step 1 for both of the illustrated embodiments, a CORE(COunterselectable REporter) cassette with KlURA3 (counterselectable)and kanMX4 (reporter) is inserted by standard DNA targeting proceduresat a DNA sequence. In these embodiments, the insertion site is anywherein the sequence that has been chosen to be deleted or is close to a sitewhere a specific mutation is to be created.

[0013] In Step 2, transformation of cells that contain the CORE-cassettewith IROs leads to loss of the CORE-cassette and (1) deletion of thedesired region or (2) introduction of the desired mutation (*). GenericDNA sequences are indicated as stippled or striped boxes. In theillustrated embodiments, the IROs have a short overlap.

[0014]FIG. 2 illustrates several specific changes produced in theindicated sequences by transformation with IROs.

[0015] Target loci within MLP2 (A), POL30 (B), TRP5 (C), SIR2 (D, E),RAD50 (F), and MREll (G) are shown, together with their wild-typenucleotide and amino acid sequence. Asterisks (*) indicate stop codons.DNA sequence changes and the corresponding amino acid substitutions areshown in boldface below the arrows. Vertical dashed lines indicate theposition in each sequence at which the CORE-cassette was integrated tocarry out the indicated mutation. The small numbers above each wild-typeDNA sequence correspond to the nucleotide position relative to the placeat which the CORE-cassette was integrated.

[0016]FIG. 3 shows the structure of several types of IROs used singly oras pairs to produce in vivo mutations as described in the examples, aswell as indicating the relative transformation efficiency of theillustrated systems.

[0017] IROs a and b overlap for 20 bases at their 3′ ends, while c and doverlap for 20 bases at their 5′ ends; e and f, e1 and f1, and e2 and f2overlap completely. IRO pairs e+j or f+i do not overlap each other.IRO-nucleotide lengths are shown on the left.

[0018] The horizontal dotted lines indicate in vitro extension of thea+b oligonucleotide pair with Pfx polymerase. The vertical dashed lineseparates sequences within IROs that are homologous to the upstream orthe downstream regions of the CORE-cassette integration point,respectively (and thus indicates where the CORE-cassette existed priorto its loss).

[0019] A qualitative summary of the transformation efficiencies arepresented to the right of the various IROs; ++, +and −, indicate high,moderate or very low efficiency, respectively.

[0020]FIG. 4 shows the specific frequencies of 5-FOA^(R)G418⁵ clones per10⁷ cells in transformation experiments with single IROs (A) or withpairs of IROs (G) in the strain BY4742-TRP5-CORE and the isogenic rad52strain. Vertical bars represent the 95% confidence interval values from3 to 6 determinations.

[0021] The types of IROs analyzed are indicated below each bar (IROs areas illustrated in FIG. 3). Pfx indicates that the oligonucleotides weresubjected to in vitro extension prior to transformation, and rad52indicates results obtained with the rad52 strain. The amount of eacholigonucleotide used was 0.5 nmoles, unless otherwise indicated (2×or10×). Beneath each bar is the mean value of the number of 5-FOA^(R)G418⁵clones/10⁷ cells followed by the mean value of the Trp⁺ clones andfinally by the % Trp⁺ among the 5-FOA^(R)G418⁵ clones. Abbreviations:n.c., negative control (no IRO DNA); NA, not applicable.

[0022] Among the G418⁵ integrants the proportion of Trp⁺ clones washigh, indicating that additional TRP5 inactivating point mutations wereinfrequent. Five random Trp⁺ clones for each type of IRO used weretested and shown to contain the expected BamHI bands, demonstrating thatthe desired mutation was successfully inserted to the TRP5 gene. Of nineTrp⁻G418₅ clones randomly chosen for sequencing, all had a frameshiftmutation confined into the IRO region.

[0023]FIG. 5 shows schematic representations of three embodiments of thedelitto perfetto mutagenesis strategy, illustrating some of theversatility of the technique for rapidly producing a variety ofmutations.

[0024]FIG. 5A shows an embodiment in which complementary 95-mers e+fhave a central region of 15 nt that can be used for site-directedmutations. 40 nt at each end of the IROs (upstream and downstream of theCORE-cassette integration site) remain unchanged in certain embodimentsfor efficient homologous integration.

[0025]FIG. 5B shows an embodiment in which 80-mers a+b filled in withPfx have a 60 nt region that can be mutagenized. Of these 60 nt, 20 ntto each side of the overlap also can be designed for random mutagenesis.As in FIG. 5A, 40 nt at each end remain unchanged in certain embodimentsfor efficient homologous integration.

[0026]FIG. 5C illustrates an embodiment in which site-specific mutationscan be introduced up to 100 bp upstream or downstream of theCORE-cassette integration point with different pairs of 80-100-mers(referred to here as m+n Pfx, analogous to a+b Pfx or to e+f), withoutmoving the integrated CORE-cassette.

[0027] The drawing shows an example of oligonucleotides designed tointroduce a mutation (*) 100 bp upstream of the CORE-cassetteintegration point with IROs m+n Pfx. The efficiency of mutagenesis maydecrease as the mutation position in the IRO sequence is placed closerto the 5′ end, since an IRO recombination event leading to excision ofthe CORE could occur without inclusion of the mutation in the IRO.

[0028]FIG. 6 illustrates an example strategy for in vivo site-specifictargeting of mutations across the DNA binding domain of p53.

[0029] Seven isogenic strains are created each with a CORE-cassetteintegrated at a different position within the DNA binding domain of p53;the construction of strain 1 (FIG. 6A), strain 2 (FIG. 6B), and strain 7(FIG. 6C) is shown.

[0030] The p53 mutations can be created simply by designingoligonucleotides that contain the desired mutation. Transformation with20 bp overlapping oligonucleotides that are homologous to a regionsurrounding the CORE-cassette results in the elimination of theCORE-cassette and creation of a site-specific change. A mutation can bedesigned 45 base pair upstream and downstream of each CORE-cassetteinsertion point. The specific illustrated embodiments show theintroduction of a mutation downstream from a CORE-cassette that wasinserted at nucleotide 315 (strain 1; FIG. 6A), nucleotide 410 (strain2; FIG. 6B) or nucleotide 885 (strain 7; FIG. 6C).

[0031]FIG. 7 illustrates an embodiment of the delitto perfetto-DSBsystem, which utilizes integrative recombinant oligonucleotides (IROs)to generate mutations.

[0032] The DSB-CORE cassette contains the COunterselectable and REportergenes plus the l-Scel endonuclease under the control of an induciblepromoter, such as the illustrated GAL1/10 promoter. Expression of -Scelleads to a double-strand break (DSB) at the unique target site, alsocontained in the cassette.

[0033] Step 1: A DSB-CORE cassette is inserted by standard DNA targetingprocedures at a desired site. The insertion site is anywhere in thesequence which has been chosen to be deleted or is close to a site wherea specific mutation, or multiple mutations, is to be created. TheDSB-CORE cassette contains the following:

[0034] i) Gal1/10 promoter fused to l-Sce open reading frame(GAL1/10::l-Scel). The l-Scel creates a DSB at a cut site included onthe cassette. Any inducible (ie., on/off) promoter can be used and anyDSB site-specific cutting enzyme can be included in the DSB-COREcassette, provided that the only DNA site that is cut in the cell islocated in the cassette.

[0035] ii) l-Scel cut site (or other unique cut site that is the targetof a DSB site-specific cutting enzyme) located at one end of thecassette or internally.

[0036] iii) COunterselectable REporter genes (i.e., thecounterselectable gene Kl-URA3 +the kanMX4 reporter gene).

[0037] Step 2: Immediately prior to transformation with IRO(s), cellsare grown in the presence of galactose, which induces l-Scelendonuclease expression. The enzyme targets its single site in thecassette and generates a DSB.

[0038] Step 3: Transformation of cells with IROs leads to loss of thecassette, creation of the desired mutation(s), or deletion of thedesired region. Oligonucleotide targeting in the vicinity of the DSB(delitto perfetto-DSB method) is increased up to 1000-fold, compared tooligo-mediated changes without the DSB (delitto perfetto method).

[0039]FIG. 8 shows schematic representations of the embodiment of thedelitto perfetto-DSB mutagenesis strategy, illustrating some of theversatility of the technique for rapidly producing a variety ofmutations.

[0040] The drawing shows an example of oligonucleotides designed tointroduce a mutation (*) 100 bp upstream of the CORE-DSB integrationpoint with IROs m+n Pfx. RE-DSB can be used in place of CORE-DSB. Theefficiency of mutagenesis by IROs replacing CORE-DSB or RE-DSB after DSBinduction is up to 1000 fold higher than when there is no DSB (i e.,when just the CORE cassette is used).

[0041]FIG. 9 illustrates an example strategy for in vivo site-specifictargeting of mutations across the DNA binding domain of p53 using theCORE-DSB. RE-DSB can be used in place of CORE-DSB. Since the efficiencyof IROs targeting using the delitto perfetto-DSB approach is veryefficient, the CORE-DSB or RE-DSB cassettes can be placed at longerintervals: up to 200 bp from one another.

[0042] Five isogenic strains are created each with a CORE-DSB or RE-DSBcassette integrated at a different position within the DNA bindingdomain of p53; the construction of strain 1 (FIG. 9A), strain 2 (FIG.9B), and strain 4 (FIG. 9C) is shown.

[0043] The p53 mutations can be created simply by designingoligonucleotides that contain the desired mutation. Transformation with20 bp overlapping oligonucleotides that are homologous to a regionsurrounding the CORE-DSB or RE-DSB cassette results in the eliminationof the cassette and creation of a site-specific change. A mutation canbe designed 100 base pair upstream and downstream of each CORE-cassetteinsertion point. The specific illustrated embodiments show theintroduction of a mutation upstream from a CORE-DSB cassette that wasinserted at nucleotide 315 (strain 1; FIG. 9A), nucleotide 515 (strain2; FIG. 9B) or nucleotide 915 (strain 4; FIG. 9C).

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0044] The nucleic and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases as defined in 37 C.F.R. 1.822. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand. In theaccompanying sequence listing:

[0045] SEQ ID NOs: 1 and 2 are the sequences used for amplification ofthe CORE cassette (the kanMX4 side and the KlURA3 side, respectively) inExample 1.

[0046] SEQ ID NOs: 3-12 are oligonucleotide primers used in initialinsertion of the CORE cassette into genes MLP2, POL30, TRP5 and SIR2. Inthe text, these sequences are referred to as MLP2.G (SEQ ID NO: 3);MLP2.U (SEQ ID NO: 4); PCNA.G (SEQ ID NO: 5); PCNA.U (SEQ ID NO: 6);TRP5.G (SEQ ID NO: 7); TRP5.U (SEQ ID NO: 8); SIR2.G1 (SEQ ID NO: 9);SIR2.U1 (SEQ ID NO: 10); SIR2.G2 (SEQ ID NO: 11); and SIR2.U2 (SEQ IDNO: 12).

[0047] SEQ ID NO: 13 is a K2 internal primer in the CORE cassette,within kanMX4.

[0048] SEQ ID NO: 14 is an URA3.2 internal primer in the CORE cassette,within KlURA3.

[0049] SEQ ID NOs: 15-26 are primers used to verify CORE insertion. Inthe text, these sequences are referred to as MLP2.1 (SEQ ID NO: 15);MLP2.2 (SEQ ID NO: 16); MLP2.3 (SEQ ID NO: 17); MLP2.4 (SEQ ID NO: 18);PCNA.3 (SEQ ID NO: 19); PCNA.4 (SEQ ID NO: 20); TRP5.1 (SEQ ID NO: 21);TRP5.2 (SEQ ID NO: 22); SIR2.1 (SEQ ID NO: 23); SIR2.2 (SEQ ID NO: 24);SIR2.3 (SEQ ID NO: 25); and SIR2.4 (SEQ ID NO: 26).

[0050] SEQ ID NOs: 27-50 are IROs used to make the specific changesdescribed in the examples In the text, these sequences are referred toas MLP2.a (SEQ ID NO: 27); MLP2.b (SEQ ID NO: 28); PCNA.a (SEQ ID NO:29); PCNA.b (SEQ ID NO: 30); TRP5.a (SEQ ID NO: 31); TRP5.b (SEQ ID NO:32); TRP5.c (SEQ ID NO: 33); TRP5.d (SEQ ID NO: 34); TRP5.e (SEQ ID NO:35); TRP5.f (SEQ ID NO: 36); TRP5.el (SEQ ID NO: 37); TRP5.fl (SEQ IDNO: 38); TRP5.e2 (SEQ ID NO: 39); TRP5.f2 (SEQ ID NO: 40); TRP5.i (SEQID NO: 41); TRP5.j (SEQ ID NO: 42); 270a (SEQ ID NO: 43); 270b (SEQ IDNO: 44); 270e (SEQ ID NO: 45); 270f (SEQ ID NO: 46); 345a (SEQ ID NO:47); 345b (SEQ ID NO: 48); 364a (SEQ ID NO: 49); and 364b (SEQ ID NO:50).

[0051] SEQ ID NOs: 51-54 are oligonucleotide primers used to verifydeletion of RAD52: RAD52.1/LEU2.2 and RAD52.4/LEU2.1. In the text, thesesequences are referred to as RAD52.1 (SEQ ID NO: 51); RAD52.4 (SEQ IDNO: 52); LEU2.1 (SEQ ID NO: 53); and LEU2.2 (SEQ ID NO: 54).

[0052] SEQ ID NOs: 55-58 are oligonucleotides (IROs) used to createlarge chromosomal deletion (up to 16 kb) around the TRP5 locus onchromosome VII. In the text, these sequences are referred to as 1Stu.a(SEQ ID NO: 55); 1Stu.b (SEQ ID NO: 56); 2Stu.a (SEQ ID NO: 57); and2Stu.b (SEQ ID NO: 58).

[0053] SEQ ID NOs: 59 and 60 are oligonucleotides that were used tocheck the large deletions. In the text, these sequences are referred toas CGR1.1 (SEQ ID NO: 59) and STT3.1 (SEQ ID NO: 60).

[0054] SEQ ID NOs: 61 and 62 are primers that were used to performinsertion of CORE cassettes into target genes. In the text, thesesequences are referred to as TRP5.1 (SEQ ID NO: 61) and TRP5.11 (SEQ IDNO: 62).

[0055] SEQ ID NOs: 63 and 64 are primers used for PCR amplification ofthe l-Scel gene under the GAL1/10 promoter; both primers contain a Bg/IIsite at the 5′ end to enable cloning of the PCR product upstream of theCORE cassettes.

[0056] SEQ ID NOs: 65-68 are oligonucleotide primers used for insertionof CORE-DSB cassettes into the TRP5 gene. In the text, these sequencesare referred to as TRP5.SceII (SEQ ID NO: 65); TSce.IU (SEQ ID NO: 66);TecS.IU (SEQ ID NO: 67); and TScecS.II (SEQ ID NO: 68).

[0057] SEQ ID NO: 69 is the GAL1/10 sequence inserted upstream from thel-Scel gene in specific embodiments.

[0058] SEQ ID NOs: 70-73 are primers used for initial insertion of theshort CORE-DSB cassette into the TRP5 gene. In the text, these sequencesare referred to as TRP5.IK (SEQ ID NO: 70); TSce.IK (SEQ ID NO: 71);TecS.IK (SEQ ID NO: 72); and TScecS.II (SEQ ID NO: 73).

[0059] SEQ ID NOs: 74-77 are internal primers in the CORE-UK and/orCORE-UH and/or CORE-Kp53 and/or CORE-Hp53 cassettes. In the text, thesesequences are referred to as K1 (SEQ ID NO: 74); URA3.1(SEQ ID NO: 75);H1 (SEQ ID NO: 76); and p7 (SEQ ID NO: 77).

[0060] SEQ ID NO: 78 is an internal primer in the CORE-DSB cassettes,within l-Scel.

[0061] SEQ ID NO: 79 is an internal primer in the RE-DSB cassettes,within HygroR.

[0062] SEQ ID NOs: 80-83 are primers that were used to delete theCORE-DSB insertion at the TRP5 locus and generate the desired change. Inthe text, these sequences are referred to as TRP5.e3 (SEQ ID NO: 80);TRP5.f3 (SEQ ID NO: 81); TRP5.e4 (SEQ ID NO: 82); and TRP5.f4 (SEQ IDNO: 83).

Detailed Description

[0063] I. Abbreviations

[0064] 5-FOA: 5-fluoroorotic acid

[0065] CORE: cassette containing a COunterselectable marker and aREporter gene

[0066] CORE-DSB: CORE cassette additionally containing a unique DSB cutsite and a sequence encoding an endonuclease that can cut at the DSB cutsite.

[0067] RE-DSB: CORE-DSB lacking the COunterselectable marker.

[0068] DNA: deoxyribonucleic acid

[0069] DSB: double strand break

[0070] G418^(R): geneticin resistance marker gene

[0071] GAL: galactose

[0072] HygroR: hygromicin resistance marker gene

[0073] IROs: integrative recombinant oligonucleotides

[0074] l-Scel: Saccharornyces cerevisiae mitochondrial intron-encodedendonuclease gene

[0075] PCR: polymerase chain reaction

[0076] ORF: open reading frame

[0077] YAC: yeast artificial chromosome

[0078] II. Terms

[0079] Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

[0080] In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

[0081] Analog, derivative or mimetic: An analog is a molecule thatdiffers in chemical structure from a parent compound, for example ahomolog (differing by an increment in the chemical structure, such as adifference in the length of an alkyl chain), a molecular fragment, astructure that differs by one or more functional groups, and/or a changein ionization. Structural analogs are often found using quantitativestructure activity relationships (QSAR), with techniques such as thosedisclosed in Remington (The Science and Practice of Pharmacology, 19thEdition (1995), chapter 28). A derivative is a biologically activemolecule derived from the base structure. A mimetic is a biomoleculethat mimics the activity of another biologically active molecule.Biologically active molecules can include both chemical structures (forinstance peptides or protein entities) that mimic the biologicalactivities of given compounds.

[0082] Animal: Living multi-cellular vertebrate organisms, a categorythat includes, for example, mammals (eg., primates) and birds as well assingle cellular and microscopic animals (e.g., nematodes). The termmammal includes both human and non-human mammals. Similarly, the term“subject” includes both human and veterinary subjects.

[0083] Antisense, sense, and antigene: Double-stranded DNA (dsDNA) hastwo strands, a 5′→3′ strand (plus strand) and a 3′→5′ strand (minusstrand). Because RNA polymerase adds nucleic acids in a 5′→3′ direction,the minus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that thebase uracil is substituted for thymine).

[0084] Antisense molecules are molecules that are specificallyhybridizable or specifically complementary to either RNA or the plusstrand of DNA. Sense molecules are molecules that are specificallyhybridizable or specifically complementary to the minus strand of DNA.Antigene molecules are either antisense or sense molecules directed to aDNA target.

[0085] Binding/stable binding: An oligonucleotide binds or stably bindsto a target nucleic acid if a sufficient amount of the oligonucleotideforms base pairs or is hybridized to its target nucleic acid, to permitdetection of that binding. Binding can be detected by physical orfunctional properties of the target:oligonucleotide complex. Bindingbetween a target and an oligonucleotide can be detected by any methodknown to one skilled in the art, including functional and physicalbinding assays. Binding can be detected functionally by determiningwhether binding has an observable effect upon a biosynthetic processsuch as expression of a gene, DNA replication, transcription, andtranslation.

[0086] Physical methods of detecting the binding of complementarystrands of DNA or RNA are well known in the art, and include suchmethods as DNase I or chemical footprinting, gel shift and affinitycleavage assays, Northern blotting, dot blotting and light absorptiondetection procedures. For example, a method that is widely used, becauseit is simple and reliable, involves observing a change in lightabsorption of a solution containing an oligonucleotide (or an analog)and a target nucleic acid at 220 to 300 nm as the temperature is slowlyincreased. If the oligonucleotide or analog has bound to its target,there is a sudden increase in absorption at a characteristic temperatureas the oligonucleotide (or analog) and target dissociate from each otheror “melt.”

[0087] The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of theoligomer is melted from its target. A higher T_(m) means a stronger ormore stable complex relative to a complex with a lower T_(m).

[0088] Cassette: A nucleic acid sequence encoding at least oneselectable marker that can be inserted into the genome of a cell or intoa plasmid, for instance a prokaryotic or eukaryotic cell. In oneembodiment, the cassette includes a reporter gene such a nucleic acidsequence that confers resistance to an antibiotic in a host cell inwhich the nucleic acid is translated. Examples of antibiotic resistancegenes include, but are not limited to, genes that provide resistance to:kanamycin, ampicillin, tetracycline, chloramphenicol, neomycin,hygromycin, and zeocin.

[0089] The most commonly used yeast genetic markers include URA3, LYS2,TRPI, LEU2, HIS3, ADE2, and G418^(R). Less frequently used yeast geneticmarkers include CYH2^(S) and CAN1^(S) (determining sensitivity tocycloheximide and canavanine, respectively); KlURA3 (from Kluyveromyceslactis and homologous to S. cerevisiae URA3, both of which determineresistance to 5-FOA); hygromicinB^(R) (determining resistance tohygromicin); and NAT^(R) (Nourseothricin) (determining resistance tonourseothricin).

[0090] Counterselectable markers (markers for which there is a systemwhere loss of the marker can be selected for) in yeast include URA3,KlURA3, CYH2, CAN1, TRP1, and LYS2. In certain embodiments,counterselectable markers URA3 and KlURA3 are particularly beneficialbecause the majority of yeast strains have a mutation in the URA3 gene(ura⁻-strains), and the frequency of spontaneous reversions is low.KlURA3 is preferred to URA3 because it can substitute URA3 of S.cerevisiae, but it is at the same time divergent enough to reduce thepossibility of gene conversion with the chromosomal mutated copy of URA3in ura⁻-strains.

[0091] Other counterselectable markers are toxic gene products that,when expressed or overexpressed prevent growth and/or kill the hostcell. Included in this class of counterselectable markers arerestriction enzymes such as EcoRI (Lewis et al., Mol Cell. Biol. 18:1891-1902, 1998) and PvuII, and the gene that encodes p53 and toxicversions of the p53 gene (Inga and Resnick, Oncogene 20: 3409-3419,2001) from humans and other mammals. These counterselectable genes aregenerally used under a highly regulatable promoter (that provides a lowbasal level and a high inducible level). In some embodiments, theexpressed PvuII gene may have modifications either in the codingsequence or in a GALl or other inducible promoter used to driveexpression of the gene. These are each examples of markers that canprovide counterselection in a broad range of biological systems forwhich more conventional counterselectable markers may not be availableor are inconvenient. These counterselectable markers are thus considered“universal” or “generic,” in that they are not dependent (or are onlyindirectly or minimally dependent) on the species or genetic backgroundof the host cell.

[0092] The following markers are also considered heterologous markers inyeast, since the involved genetic sequence is not native to S.cerevisiae but has been added from a different species: KlURA3,G418^(R), hygromicin^(R), NAT^(R), and p53.

[0093] A specific example of a cassette is a CORE-cassette, whichcontains both a COunterselectable marker and a REporter gene. Otherspecific examples include RE-cassettes (having a reporter gene but nocounter selectable marker), CORE-DSB-cassettes (having acounterselectable marker, a reporter gene, a double-strand break site,and a sequence encoding a double-strand break enzyme having specificactivity for that site), and RE-DSB-cassettes (similar toCORE-DSB-cassettes, but lacking a counterselectable marker). Alsocontemplated are cassettes for use in delitto perfetto-DSB mutagenesis,wherein the function of the double-strand break enzyme is not encoded bythe cassette itself, but is instead added exogenously to the cells,usually after insertion of the cassette and prior to (or concurrentwith) transformation with IRO(s). cDNA (complementary DNA): A piece ofDNA lacking internal, non-coding segments (introns) and regulatorysequences which determine transcription. cDNA may be synthesized in thelaboratory by reverse transcription from messenger RNA extracted fromcells.

[0094] Deletion: The removal of a sequence of DNA, the regions on eitherside of the removed sequence being joined together. Correspondingly, adeletion in a protein is the removal of a region of amino acid sequenceof the protein or peptide. Deletions can be quite short, for instanceonly one or a few nucleic acids to 10, 15, 20, 25, 30, 50, 80, or 100nucleic acids or longer, and may be quite long. In particularembodiments long deletions may be at least 500 nucleic acids, at least750, at least 1000, at least 2500, at least 3000, at least 5000, atleast 8000, at least 10,000, or more nucleic acids in length.Particularly long deletions may be over 10,000 nucleic acids, forinstance as long as 15,000, 20,000, 30,000, or more.

[0095] DNA (deoxyribonucleic acid): DNA is a long chain polymer thatcomprises the genetic material of most living organisms (some viruseshave genes comprising ribonucleic acid, RNA). The repeating units in DNApolymers are four different nucleotides, each of which comprises one ofthe four bases, adenine, guanine, cytosine, and thymine bound to adeoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides, referred to as codons, in DNA molecules code for amino acidin a polypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

[0096] Unless otherwise specified, any reference to a DNA molecule isintended to include the reverse complement of that DNA molecule. Exceptwhere single-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule.

[0097] Double strand break: Breaks that occur in the DNA backbones ofboth strands at approximately the same nucleotide pair are calleddouble-strand breaks.

[0098] Endonuclease: An enzyme that breaks the internal phosphodiesterbonds in a DNA molecule.

[0099] Heterologous: A sequence that is not normally (i.e., in thewild-type sequence) found adjacent to a second sequence. In oneembodiment, the sequence is from a different genetic source, such as avirus or other organism, than the second sequence.

[0100] Homing endonucleases: Site-specific endonucleases encoded byintron or intein to promote homing of a genetic element into theintronless or inteinless allele. They are double stranded DNAses thathave large, asymmetric recognition site (12-40 base pairs) and codingsequences that are usually embedded in their introns or inteins. Intronsare spliced out of precursor RNAs, while inteins are spliced out ofprecursor proteins. Homing endonucleases are named using conventionssimilar to those of restriction endonucleases, with intron encodingendonucleases containing the prefix, “I-” and intein endonucleasescontaining the prefix, “PI-”.

[0101] Homing endonuclease recognition sites are extremely rare. Forexample, an 18 base pair recognition will occur only once in every7×10¹⁰ base pairs of random sequence. This is equivalent to only onesite in 20 mammalian-sized genomes. However, unlike standard restrictionendonucleases, homing endonucleases tolerate some sequence degeneracywithin their recognition sequence. As a result, their observed sequencespecificity is typically in the range of 10-12 base pairs. Homingendonucleases do not have stringently-defined recognition sequences inthe way that restriction enzymes do. That is, single base changes do notabolish cleavage but reduce its efficiency to variable extents. Theprecise boundary of required basis is generally not known.

[0102] The homing enzymes that initiate the mobility process can begrouped into families, which share structural and functional propertieswith each other and with some freestanding, intergenic endonucleases.Several endonucleases encoded by introns and inteins in the threebiological kingdoms have been shown to promote the homing of theirrespective genetic elements into allelic intronless and inteinless sites(Belfort & Roberts, Nucleic Acids Research, 25: 3379-3388, 1997).Examples of homing endonuclease: I-CreI, I-PpoI, I-SceI, I-CeuI,PI-PspI, PI-SceI. Among homing endonucleases there is also VDE ofSaccharomyces.

[0103] Hybridization: Poly- and oligonucleotides and their analogshybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary bases.Generally, nucleic acid consists of nitrogenous bases that are eitherpyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines(adenine (A) and guanine (G)). These nitrogenous bases form hydrogenbonds between a pyrimidine and a purine, and the bonding of thepyrimidine to the purine is referred to as “base pairing.” Morespecifically, A will hydrogen bond to T or U, and G will bond to C.“Complementary” refers to the base pairing that occurs between twodistinct nucleic acid sequences or two distinct regions of the samenucleic acid sequence. “Specifically hybridizable” and “specificallycomplementary” are terms that indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between thepoly- or oligonucleotide (or its analog) and the DNA or RNA target. Thepoly- or oligonucleotide (or its analog) need not be 100% complementaryto its target sequence to be specifically hybridizable. A nucleotidemolecule or analog thereof is specifically hybridizable when its bindingto a target DNA or RNA molecule occurs with a sufficient degree ofcomplementarity to avoid non-specific binding of the nucleotide oranalog to non-target sequences under conditions where specific bindingis desired, for example under physiological conditions in the case of invivo assays or systems. Such binding is referred to as specifichybridization.

[0104] Hybridization conditions resulting in particular degrees ofstringency will vary depending on the nature of the hybridization methodof choice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ concentration) of the hybridization bufferwill determine the stringency of hybridization, though wash times alsoinfluence stringency. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, chapters 9 and 11, incorporated herein by reference.

[0105] By way of illustration, hybridization is generally carried out invitro in a solution of high ionic strength such as 6×SSC at atemperature that is 20-25° C. below the melting temperature, T_(m),described below. For instance, for Southern hybridization experimentswhere the target DNA molecule on the Southern blot contains 10 ng of DNAor more, hybridization is typically carried out for 6-8 hours using 1-2ng/ml radiolabeled poly- or oligonucleotide probe (of specific activityequal to 10⁹ CPM/μg or greater, for instance). Following hybridization,the nitrocellulose filter (Southern blot) is washed to remove backgroundhybridization. The washing conditions should be as stringent as possibleto remove background hybridization but to retain a specifichybridization signal.

[0106] The term T_(m) represents the temperature above which, under theprevailing ionic conditions, the probe nucleic acid molecule will nothybridize to its target DNA molecule. The T_(m) of such a hybridmolecule may be estimated from the following equation:

T _(m)=81.5° C.−16.6(log₁₀[Na^(+])+)0.41(% G+C)−0.63 (%formamide)−(600/l)

[0107] Where l=the length of the hybrid in base pairs.

[0108] This equation is valid for concentrations of Na⁺ in the range of0.01 M to 0.4 M, and it is less accurate for calculations of T_(m) insolutions of higher [Na⁺]. The equation is also primarily valid for DNAswhose G+C content is in the range of 30% to 75%, and it applies tohybrids greater than 100 nucleotides in length (the behavior ofoligonucleotide probes is described in detail in Ch. 11 of Sambrook etal., 1989). Thus, by way of example, for a 150 base pair DNA probe witha hypothetical GC content of 45%, a calculation of hybridizationconditions required to give particular stringencies may be made asfollows:

[0109] For this example, it is assumed that the filter will be washed in0.3×SSC solution following hybridization, thereby

[0110] [Na⁺]=0.045M

[0111] % GC=45%

[0112] Formamide concentration=0

[0113] l=150 base pairs

[0114] T_(m)=81.5−16(log₁₀[Na⁺])+(0.41×45)−(600/150)

[0115] and so T_(m)=74.4° C.

[0116] The T_(m) of double-stranded DNA decreases by 1-1.5° C. withevery 1% decrease in homology (Bonner et al., J. Mol. Biol. 81:123-135,1973). Therefore, for this given example, washing the filter in 0.3×SSCat 59.4-64.4° C. will produce a stringency of hybridization equivalentto 90%; that is, DNA molecules with more than 10% sequence variationrelative to the target cDNA will not hybridize. Alternatively, washingthe hybridized filter in 0.3×SSC at a temperature of 65.4-68.4° C. willyield a hybridization stringency of 94%; that is, DNA molecules withmore than 6% sequence variation relative to the target cDNA moleculewill not hybridize. The above examples are given entirely by way oftheoretical illustration. One skilled in the art will appreciate thatother hybridization techniques may be utilized and that variations inexperimental conditions will necessitate alternative calculations forstringency.

[0117] For purposes of the present invention, “stringent conditions”encompass conditions under which hybridization will only occur if thereis less than 25% mismatch between the hybridization probe and the targetsequence. “Stringent conditions” may be broken down into particularlevels of stringency for more precise distinction. Thus, as used herein,“moderately stringent” conditions are those under which DNA moleculeswith more than 25% sequence variation (also termed “mismatch”) will nothybridize; “medium stringent” conditions are those under which DNAmolecules with more than 15% mismatch will not hybridize, and “highlystringent” conditions are those under which DNA sequences with more than10% mismatch will not hybridize. “Very highly stringent” conditions arethose under which DNA sequences with more than 6% mismatch will nothybridize.

[0118] In vitro amplification: Techniques that increase the number ofcopies of a nucleic acid molecule in a sample or specimen. An example ofin vitro amplification is the polymerase chain reaction (PCR), in whicha nucleic acid molecule (such as one contained in a biological samplecollected from a subject) is contacted with a pair of oligonucleotideprimers, under conditions that allow for the hybridization of theprimers to nucleic acid template in the sample. The primers are extendedunder suitable conditions, dissociated from the template, and thenre-annealed, extended, and dissociated to amplify the number of copiesof the nucleic acid.

[0119] The product of in vitro amplification may be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing, using standard techniques.

[0120] Other examples of in vitro amplification techniques includestrand displacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see WO 90/01069);ligase chain reaction amplification (see EP-A-320 308); gap fillingligase chain reaction amplification (see U.S. Pat. No. 5,427,930);coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); andNASBA™ RNA transcription-free amplification (see U.S. Pat. No.6,025,134).

[0121] Inducer: A small molecule that triggers gene transcription bybinding to a regulator protein.

[0122] Inducible promoter: A promoter system that can be used toregulate the amount and the timing of protein expression. Unlikeconstitutive promoters, an inducible promoter is not always active. Someinducible promoters are activated by physical stimuli, such as the heatshock promoter. Others are activated by chemical stimuli, such as IPTGor Tetracycline (Tet), or galactose. Inducible promoters orgene-switches are used to both spatially and temporally regulate geneexpression

[0123] By allowing the time and/or location of gene expression to beprecisely regulated, gene-switches or inducible promoters may controldeleterious and/or abnormal effects caused by overexpression ornon-localized gene expression. Thus, for a typical inducible promoter inthe absence of the inducer, there would be little or no gene expressionwhile, in the presence of the inducer, expression should be high (i.e.,off/on). A number of controllable gene expression systems have beendevised, including those regulated by heat (Ainley and Key, Plant Mol.Biol., 14:949-967, 1990; Holtorf et al., Plant Mol. Biol. 29:637-646,1995), pathogens (PR1-a; Williams et al., Biotechnology 10:540-543,1992; Gatz, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108, 1997),herbicide safeners (In2-2, GST-27; De Veylder et al., Plant CellPhysiol. 38:568-577, 1997), light (Kuhlemeier et al., Plant Cell1:471-478, 1989), wounding (Firek et al., Plant Mol. Biol. 22:129-212,1993), ethanol (Salter et al., Plant J. 16:127-132, 1998), phytohormones(Li et al., Plant Cell 3:1167-1175, 1991), steroids (Aoyama and Chua,Plant J., 11:605-612, 1997), and tetracycline (Gatz et al., Plant J2:397-404, 1992; Weinmann et al., Plant J., 5:559-569, 1994; Sommer etal., Plant Cell Rep. 17:891-896, 1998)(from Granger & Cyr, Plant CellReports 20:227-234, 2001).

[0124] The following are examples of specific types of induciblepromoters and examples of their use:

[0125] Steroid inducible expression: Keith Yamamoto's lab developed aninducible system in yeast similar to the ecdysone system we talked aboutfor mammalian cells. They placed the rat glucocorticoid receptor genebehind the constitutive GPD promoter to express the rat glucocorticoidreceptor in yeast. A second vector was made with three glucocorticoidresponse elements upstream of the CYCl gene minimal promoter (cytochromec gene). A cloning site was placed after this so a selected gene couldbe placed under control of the 3GRE/CYCl promoter. Both vectors werehigh copy vectors. This system works well with dose dependent expressionwhen steroid hormone is added to the medium. Response time is rapid witht_(1/2) of 7-9 minutes after addition of hormone.

[0126] Copper inducible expression: The CUP1 promoter can be used tomake a gene inducible by copper or silver ions. By way of example, agene, when placed under CUP1 regulation, should e provided with a degreeof control of the level of expression based on the amount of copper inthe medium. Copper is toxic and any strain should be tested to see howwell it tolerates copper before making a CUP1 construct.

[0127] Heat shock expression: By placing the UAS from a heat shock genein front of the minimal CYCl promoter, you can place YFG (your favoritegene) under heat shock induction. This is a specialized requirementusually used in studies of heat shock response GAL1/10 promoter: Thispromoter is highly regulatable by galactose, such that there is a basallevel on glucose, but over 100 fold increase when cells are placed ingalactose medium.

[0128] The yeast GAL genes form one of the most intensely studied modelsystems for eukaryotic gene regulation. The structural genes, e.g. GAL1and GAL10, are induced to very high level expression in galactose by theaction of the activator Gal4p. Gal4p binds to activation sequences(UASG) that lie up stream of GAL genes and activates transcription in aprocess that depends on gene-proximal TATA elements and involvesnumerous coactivators and general transcription factors including TBP.The activation function of Gal4p is modulated by Gal80p, an inhibitoryregulator that binds specifically to the activation domain of Gal4p,thus preventing gene activation in nongalactose carbon sources.

[0129] Induction: The ability of a bacteria (or yeast, or otherorganism) to synthesize certain enzymes only when their substrates arepresent; applied to gene expression refers to switching on transcriptionas a result of interaction of the inducer with the regulator protein.

[0130] Isolated: An isolated biological component (such as a nucleicacid, peptide or protein) has been substantially separated, producedapart from, or purified away from other biological components in thecell of the organism in which the component naturally occurs, i.e.,other chromosomal and extra-chromosomal DNA and RNA, and proteins.Nucleic acids, peptides, and proteins that have been isolated includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

[0131] Nucleic acid: A sequence composed of nucleotides, including thenucleotides that are found in DNA and RNA.

[0132] Nucleotide: This term includes, but is not limited to, a monomerthat includes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

[0133] Oligonucleotide: A linear polynucleotide sequence usually of upto about 200 nucleotide bases in length, for example a polynucleotide(such as DNA or RNA) which is at least six nucleotides, for example atleast 15, 20, 50, 100 or even 200 nucleotides long. In certainembodiments, it is envisioned that oligonucleotides may be over 200nucleotides in length, for instance, 220, 250, 270, 290, 300, 350, 400or more nucleotides.

[0134] Operably linked: A first nucleic acid sequence is operably linkedwith a second nucleic acid sequence when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

[0135] ORF (open reading frame): A series of nucleotide triplets(codons) coding for amino acids. These sequences are usuallytranslatable into a peptide.

[0136] Ortholog: Two nucleotide sequences are orthologs of each other ifthey share a common ancestral sequence, and diverged when a speciescarrying that ancestral sequence split into two species. Orthologoussequences are also homologous sequences.

[0137] Polynucleotide: A linear nucleic acid sequence of any length.Therefore, a polynucleotide includes molecules which are at least 15,20, 50, 100, 200, 250, 300, 400 (e.g., oligonucleotides)or more, andalso including nucleotides as long as a full length cDNAs, genes, orchromosomes.

[0138] Peptide Nucleic Acid (PNA): An oligonucleotide analog with abackbone comprised of monomers coupled by amide (peptide) bonds, such asamino acid monomers joined by peptide bonds.

[0139] Probes and primers: A probe comprises an isolated nucleic acidattached to a detectable label or reporter molecule. Typical labelsinclude radioactive isotopes, ligands, chemiluminescent agents, andenzymes. Methods for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, e.g., in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (1989); and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-lntersciences(1987).

[0140] Primers are short nucleic acids, for example DNA oligonucleotidesat least 6 nucleotides in length, and/or no longer than 10, 20, 50, 100or 200 nucleotides in length, though in some embodiments they arelonger. Primers may be annealed to a complementary target DNA strand bynucleic acid hybridization to form a hybrid between the primer and thetarget DNA strand, and then extended along the target DNA strand by aDNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, e.g., by PCR or other nucleic acid amplificationmethods known in the art.

[0141] Methods for preparing and using probes and primers are described,for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1989), Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates andWiley-Intersciences (1987), and Innis et al., PCR Protocols, A Guide toMethods and Applications, 1990, Innis et al. (eds.), 21-27, AcademicPress, Inc., San Diego, Calif. PCR primer pairs can be derived from aknown sequence, for example, by using computer programs intended forthat purpose, such as Primer (Version 0.5, © 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.).

[0142] Probes and primers disclosed herein comprise at least 10nucleotides of a nucleic acid sequence, although a shorter nucleic acidmay be used as a probe or primer if it specifically hybridizes understringent conditions with a target nucleic acid by methods well known inthe art. The disclosure thus includes isolated nucleic acid moleculesthat include specified lengths of the disclosed sequences. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer comprising20 consecutive nucleotides of a sequence will anneal to a targetsequence (for instance, contained within a genomic DNA library) with ahigher specificity than a corresponding primer of only 15 nucleotides.To enhance specificity, longer probes and primers can be used, forexample probes and primers that comprise at least 20, 30, 40, 50, 60,70, 80, 90, 100 or more consecutive nucleotides from any region of atarget.

[0143] When referring to a probe or primer, the term “specific for (atarget sequence)” indicates that the probe or primer hybridizes understringent conditions substantially only to the target sequence in agiven sample comprising the target sequence.

[0144] Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase 11 type promoter, a TATA element. In oneembodiment, a promoter includes an enhancer. In another embodiment, apromoter includes a repressor element. In these embodiments, a chimericpromoter is created (a promoter/enhancer chimera or a promoter/repressorchimera, respectively). Enhancer and repressor elements can be locatedadjacent to, or distal to the promoter, and can be located as much asseveral thousand base pairs from the start site of transcription.Examples of promoters that can be used in the present disclosureinclude, but are not limited to the SV40 promoter, the CMVenhancer-promoter, the CMV enhancer/μ-actin promoter, and thetissue-specific promoter probasin.

[0145] Other promoter sequences which can be used to construct thenucleic acids and practice the methods disclosed herein include, but arenot limited to: the lac system, the trp system, the tac system, the trcsystem, major operator and promoter regions of phage lambda, the controlregion of fd coat protein, the early and late promoters of SV40,promoters derived from polyoma, adenovirus, retrovirus, baculovirus andsimian virus, the promoter for 3-phosphoglycerate kinase, the promotersof yeast acid phosphatase, the promoter of the yeast alpha-matingfactors, any retroviral LTR promoter such as the RSV promoter; induciblepromoters, such as the MMTV promoter; the metallothionein promoter; heatshock promoters; the albumin promoter; the histone promoter; the α-actinpromoter; TK promoters; B19 parvovirus promoters; the SV10 latepromoter; the ApoAI promoter and combinations thereof.

[0146] In one embodiment, a promoter is a strong promoter, whichpromotes transcription of RNA at high levels, for example at levels suchthat the transcriptional activity of the promoter generally accounts forabout 25% of transcriptional activity of all transcription within acell. The strength of a promoter is often tissue-specific and thus mayvary from one cell type to another. For example, CMV is a classic strongpromoter because it generates high levels of transcriptional activity inmany cell types. Examples of strong promoters include, but are notlimited to: CMV; CMV/chicken β-actin; elongation factors 1A and 2A;SV40; RSV; and the MoLV LTR.

[0147] In another embodiment, a promoter is a tissue-specific promoter,which promotes transcription in a single cell type or narrow range oftissues. Examples of tissue-specific promoters include, but are notlimited to: probasin (which promotes expression in prostate cells), animmunoglobulin promoter; a whey acidic protein promoter; a caseinpromoter; glial fibrillary acidic protein promoter; albumin promoter;β-globin promoter; and the MMTV promoter.

[0148] In yet another embodiment, a promoter is a hormone-responsivepromoter, which promotes transcription only when exposed to a hormone.Examples of hormone-responsive promoters include, but are not limitedto: probasin (which is responsive to testosterone and other androgens);MMTV promoter (which is responsive to dexamethazone, estrogen, andandrogens); and the whey acidic protein promoter and casein promoter(which are responsive to estrogen).

[0149] For expression of yeast genes in yeast, there are a variety ofpromoters to choose from for various purposes. The following areprovided by way of example, and are not meant to be in any way limiting:

[0150] The Gal 1,10 promoter: This promoter is inducible by galactose.It is frequently valuable to be able to turn expression of your gene onand off so you can follow the time dependent effects of expression. TheGal promoter is slightly leaky, and so is appropriate where it is notessential to have absolutely no expression of the passenger gene in theabsence of galactose. The Gal 1 gene and Gal 10 gene are adjacent andtranscribed in opposite directions from the same promoter region. Theregulatory region containing the UAS sequences can be cut out on a DdelSau3A fragment and placed upstream of any other gene to confer galactoseinducible expression and glucose repression.

[0151] PGK, GPD and ADH1 promoters: These are high expressionconstitutive promoters. PGK=phosphoglycerate kinase, GPD=glyceraldehyde3 phosphate dehydrogenase, ADH1=alcohol dehydrogenase ADH2 promoter:This gene is glucose repressible and it is strongly transcribed onnon-fermentable carbon sources (similar to GAL 1,10 except not inducibleby galactose).

[0152] CUP1 promoter: This is the metalothionein gene promoter. It isactivated by copper or silver ions added to the medium. The CUP1 gene isone of a few yeast genes that is present in yeast in more than one copy.Depending on the strain, there can be up to eight copies of this gene.

[0153] PHO5 promoter: This promoter is derived from a gene that encodesan acid phosphatase. It is induced by low or no phosphate in the medium.The phosphatase is secreted in the chance it will be able to free upsome phosphate from the surroundings. When phosphate is present, no PHO5message can be found. When it is absent it is turned on strongly.

[0154] Protein: A biological molecule expressed by a gene and comprisedof amino acids.

[0155] Purified: The term purified does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedprotein (or nucleic acid) preparation is one in which the protein (ornucleic acid) is more pure than the molecule in its natural environmentwithin a cell (or other production vessel). In one embodiment, apreparation of a molecule is purified such that the molecule representsat least 50%, for example at least 70%, of the total content of thepreparation.

[0156] Random oligonucleotide: An oligonucleotide with a random sequence(see, for instance, U.S. Pat. Nos. 5,043,272 and 5,106,727, whichillustrate random oligonucleotides used for priming in vitroamplification reactions) in at least one position within the length ofthe oligonucleotide.

[0157] The sequences of random oligonucleotides may not be random in theabsolute mathematic sense. For instance, chemically synthesized randomoligonucleotide will be random to the extent that physical and chemicalefficiencies of the synthetic procedure will allow, and based on themethod of synthesis. Oligonucleotides having defined sequences maysatisfy the definition of random if the conditions of their use causethe locations of their apposition to the template to be indeterminate.Also, random oligonucleotides may be “random” only over a portion oftheir length, in that one residue within the sequence, or a portion ofthe sequence, can be identified and defined prior to synthesis of theprimer.

[0158] Random oligonucleotides may be generated using availableoligonucleotide synthesis procedures; randomness of the sequence may beintroduced by providing a mixture of nucleic acid residues in thereaction mixture at one or more addition steps (to produce a mixture ofoligonucleotides with random sequence at that residue position). Thus,an oligonucleotide that is random throughout its length can be generatedby sequentially incorporating nucleic acid residues from a mixture of25% of each of dATP, dCTP, dGTP, and dTTP, to form an oligonucleotide.Other ratios of dNTPs can be used (e.g., more or less of any one dNTP,with the other proportions adapted so the whole amount is 100%).

[0159] The term “random oligonucleotide” specifically includes acollection of individual oligonucleotides of different sequences, forinstance, which can be indicated by the generic formula 5′-XXXXX-3′,wherein X represents a nucleotide residue that was added to theoligonucleotide from a mixture of a definable percentage of each dNTP.For instance, if the mixture contained 25% each of dATP, dCTP, dGTP, anddTTP, the indicated oligonucleotide would contain a mixture ofoligonucleotides that have a roughly 25% average chance of having A, C,G, or T at each position.

[0160] This term also includes a mixture of oligonucleotides with thegeneric formula 5′-nnnXnn-31, wherein X is defined as in the firstformula and n denotes a known nucleotide (e.g., A, C, G, or T).

[0161] Recombinant: A recombinant nucleic acid is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques, suchas those described in Sambrook et al. (In: Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., 1989).

[0162] Restriction endonucleases: Enzymes that recognize specificnucleotide sequences and cleave both strands of the DNA containing thosesequences.

[0163] Sequence identity: The identity between two or more nucleic acidsequences, or two or more amino acid sequences, is expressed in terms ofthe identity between the sequences. Sequence identity can be measured interms of percentage identity (or similarity or homology); the higher thepercentage, the more near to identical the sequences are to each other.Homologs or orthologs of nucleic acid or amino acid sequences possess arelatively high degree of sequence identity when aligned using standardmethods. This homology is more significant when orthologous proteins orcDNAs are derived from species that are more closely related (e.g. humanand monkey sequences), compared to species more distantly related (e.g.,human and C. elegans sequences).

[0164] Methods of alignment of sequences for comparison are well knownin the art. Various programs and alignment algorithms are described in:Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, JMol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. Biosc. 8, 155-65, 1992; and Pearson et al.,Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J Mol. Biol.215:403-10, 1990, presents a detailed consideration of sequencealignment methods and homology calculations.

[0165] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul etal., J MoL Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

[0166] For comparisons of amino acid sequences of greater than about 30amino acids, the Blast 2 sequences function is employed using thedefault BLOSUM62 matrix set to default parameters, (gap existence costof 11, and a per residue gap cost of 1). When aligning short peptides(fewer than around 30 amino acids), the alignment should be performedusing the Blast 2 sequences function, employing the PAM30 matrix set todefault parameters (open gap 9, extension gap 1 penalties). Proteinswith even greater identity to the reference sequence will showincreasing percentage identities when assessed by this method, such asat least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity,when using gapped blastp with databases such as the nr, pat, orswissprot database. Queries searched with the blastp program arefiltered with DUST (Hancock and Armstrong, Comput. Appl. Biosci.10:67-70, 1994). Other programs use SEG.

[0167] When less than the entire sequence is being compared for sequenceidentity, homologs typically possess at least 75% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85%, 90%, 95% or 98% or more, depending on their identity tothe reference sequence. Methods for determining sequence identity oversuch short windows are described at the NCBI web site.

[0168] One of skill in the art will appreciate that these sequenceidentity ranges are provided for guidance only; it is entirely possiblethat significant homologs can be obtained that fall outside of theranges provided.

[0169] Site-specific endonuclease: An enzyme that generates a DSB at aspecific site within a nucleic acid molecule. By way of example, yeastHO is an endonuclease, required for mating-type switching in yeast. TheHO endonuclease cleaves the DNA sequence at MAT, the mating type locus,that determines the cell's mating type (either a or α). The DSB causes agene conversion event to occur using either of two donor sequences,called HML and HMR, leading to the unidirectional transfer of matingtype information from HML or HMR to MAT. The HO gene encodes anendonuclease that recognizes a consensus sequence of 24 bp and makes a 4bp staggered cleavage within that sequence.

[0170] Transduced and Transfected: A virus or vector transduces ortransfects a cell when it transfers nucleic acid into the cell. A cellis “transfected” by a nucleic acid transduced into the cell when the DNAbecomes stably replicated by the cell, either by incorporation of thenucleic acid into the cellular genome, or by episomal replication.

[0171] Transformed: A transformed cell is a cell into which has beenintroduced a nucleic acid molecule by molecular biology techniques. Asused herein, the term transformation encompasses all techniques by whicha nucleic acid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

[0172] Vector: A nucleic acid molecule as introduced into a host cell,thereby producing a transformed host cell. A vector may include nucleicacid sequences that permit it to replicate in a host cell, such as anorigin of replication. A vector may also include one or more selectablemarker genes and other genetic elements known in the art.

[0173] Unless otherwise explained, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs. The singularterms “a”, “an,” and “the” are intended to include plurals unlesscontext clearly indicates otherwise. It is further to be understood thatall base sizes or amino acid sizes, and all molecular weight ormolecular mass values, given for nucleic acids or polypeptides areapproximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0174] III. Overview of Several Embodiments

[0175] A first embodiment is a method for introducing a mutation into atarget double stranded nucleic acid sequence in a cell, wherein thedouble stranded nucleic acid sequence comprises a first and a secondstrand, and the method involves introducing a double-stranded nucleicacid cassette into a target nucleic acid sequence at an insertion point,wherein the cassette is a RE-cassette and comprises a first portionhomologous to a nucleic acid sequence on a first side of the insertionpoint; a second portion homologous to a second nucleic acid sequence ona second side of the insertion point; and a nucleic acid sequenceencoding a reporter located between the first portion and the secondportion; transforming the cell with a first oligonucleotide comprising anucleic acid sequence homologous to one strand (the chosen strand) ofthe target nucleic acid sequence at a position on the first side of theinsertion point; and a nucleic acid sequence homologous to the samestrand of the target nucleic acid sequence at a position on the secondside of the insertion point, and comprising at least one nucleotide thatdiffers from the chosen strand of the target nucleic acid sequence; andselecting for loss of the nucleic acid sequence encoding the reportergene, wherein loss of the nucleic acid sequence encoding the reportergene indicates integration of the oligonucleotide sequence comprisingthe at least one nucleotide that differs from the target nucleic acidsequence.

[0176] A second embodiment is a method for introducing a mutation into atarget double stranded nucleic acid sequence in a cell, wherein thedouble-stranded nucleic acid cassette further comprises a nucleic acidsequence encoding a counterselectable marker located between the firstportion and the second portion, which cassette is referred to as aCORE-cassette, and wherein the method further comprises selecting forloss of both the nucleic acid encoding the counterselectable marker andthe nucleic acid sequence encoding the reporter gene, wherein loss ofboth the nucleic acid sequence encoding the counterselectable marker andthe nucleic acid sequence encoding the reporter gene indicatesintegration of the oligonucleotide sequence comprising the at least onenucleotide that differs from the target nucleic acid sequence.Optionally the double-stranded nucleic acid cassette in certainembodiments further comprises a nucleic acid sequence comprising adouble-strand break recognition site, a nucleic acid encoding adouble-strand break enzyme that recognizes the double-strand breakrecognition site, and an inducible promoter, operably connected with thenucleic acid encoding the double-strand break enzyme, which cassette isreferred to as a CORE-DSB-cassette.

[0177] Yet another embodiment is a method for introducing a mutationinto a target double stranded nucleic acid sequence in a cell, whereinthe double-stranded nucleic acid cassette further comprises a nucleicacid sequence comprising a double-strand break recognition site, anucleic acid encoding a double-strand break enzyme that recognizes thedouble-strand break recognition site, and an inducible promoter,operably connected with the nucleic acid encoding the double-strandbreak enzyme, which cassette is referred to as a RE-DSB-cassette.

[0178] In specific embodiments that include in the cassette adouble-strand break site and nucleic acid sequence encoding adouble-strand break enzyme, the method further comprises inducingexpression of the double strand break enzyme, thereby stimulating adouble-strand break within the cassette, which double-strand breakstimulates recombination.

[0179] In specific examples of the provided methods, the oligonucleotidesequence comprises more than one nucleotide that differs from the targetnucleic acid sequence. In certain examples, transforming the cell withthe first oligonucleotide occurs prior to selecting for loss of both thenucleic acid encoding the counterselectable marker and the nucleic acidencoding the reporter gene.

[0180] Also provided are methods that further comprise transforming thecell with a second oligonucleotide (for instance, concurrently withtransforming the cell with the second oligonucleotide) that is at leastpartially complementary to the first oligonucleotide. In specificexamples, the second oligonucleotide comprises a nucleic acid sequencehomologous to the target nucleic acid sequence at a position to thefirst side of the insertion point. In other examples, the secondoligonucleotide comprises a nucleic acid sequence homologous to thetarget nucleic acid sequence at a position to the second side of theinsertion point. In still other examples, the second oligonucleotidecomprises a nucleic acid sequence homologous to the target nucleic acidsequence at a position to the first side of the insertion point; and anucleic acid sequence homologous to the target nucleic acid sequence ata position to the second side of the insertion point.

[0181] In various embodiments, the first and/or second oligonucleotidecontains at least one random nucleotide change compared to the targetnucleic acid sequence, and optionally contains several. In certainembodiments, the second oligonucleotide is fully complementary to thefirst oligonucleotide. In other embodiments, the 3′ ends of the twooligonucleotides are complementary but the first oligonucleotide lackshomology to the second side of the insertion point and the secondoligonucleotide lacks homology to the first side of the insertion point.In still other embodiments, the 3′ ends of the two oligonucleotides arecomplementary but the second oligonucleotide lacks homology to thesecond side of the insertion point and the first oligonucleotide lackshomology to the first side of the insertion point. In yet anotherembodiment, the 5′ ends of the two oligonucleotides are complementarybut the first oligonucleotide lacks homology to the second side of theinsertion point and the second oligonucleotide lacks homology to thefirst side of the insertion point. Alternatively, in certain embodimentsthe 5′ ends of the two oligonucleotides are complementary but the secondoligonucleotide lacks homology to the second side of the insertion pointand the first oligonucleotide lacks homology to the first side of theinsertion point.

[0182] Also provided are methods using CORE-cassettes orCORE-DSB-cassettes, wherein the counterselectable marker is KlURA3,URA3, TRP5, TRP1, or a gene encoding a toxin. In specific examples ofsuch methods, the counterselectable marker is a gene encoding a toxin,and the toxin is an inducible restriction enzyme or an inducible p53gene. Optionally, the inducible p53 gene is a toxic version.

[0183] In some embodiments, the reporter encodes a polypeptide thatconfers antibiotic resistance to the cell. The antibiotic is G418,hygromycin, kanamycin, ampicillin, tetracycline, chloramphenicol,neomycin, zeocin, nourseothricin, cycloheximide or canavanine inspecific examples.

[0184] Also described herein are methods wherein the reporter encodes apolypeptide from an amino acid or nucleotide synthesis pathway. Thepolypeptide is LEU2, TRP5, TRP1, LYS2, HIS3, or ADE2 in particularexamples.

[0185] The oligonucleotide in various methods can be of any length, forinstance at least 30 nucleotides, at least 40 nucleotides, at least 50nucleotides, or at least 80 nucleotides in length.

[0186] Also described herein are methods in which the first and thesecond oligonucleotides are each at least 30 nucleotides in length. Inother methods, the first and the second oligonucleotides are each atleast 40 nucleotides in length, at least 50 nucleotides in length, or atleast 80 nucleotides in length.

[0187] In certain embodiments, the first and the second oligonucleotideare of different lengths.

[0188] In certain embodiments, the region of overlap at the 3′ ends ofthe oligonucleotides is at least 10 base pairs; in others, the region ofoverlap at the 3′ ends is at least 15 base pairs. In specific examplesof such methods, the 3′ ends of the first and second oligonucleotide canbe extended by in vitro polymerization.

[0189] Also provided are methods wherein at least one oligonucleotidediffers from the target nucleic acid sequence by more than onenucleotide. In other examples, both oligonucleotides differ from thetarget nucleic acid sequence by at least a single nucleotide. Inspecific examples, at least one nucleotide difference is inside theregion of overlap between the first and second oligonucleotides, whilein other examples, at least one nucleotide difference is outside theregion of overlap between the first and second oligonucleotides. In someexamples, differences occur both inside and outside of the region ofoverlap.

[0190] Another embodiment is a method of deleting a target doublestranded nucleic acid sequence (for instance, from about 1 to up toabout 16,000 bp in length) from within a cell, wherein the target doublestranded nucleic acid sequence comprises a first and a second strand,the method comprising introducing a double-stranded nucleic acidcassette into a target nucleic acid sequence at an insertion point,wherein the cassette is a RE-cassette and comprises a nucleic acidsequence encoding a reporter located between a first portion and asecond portion of the RE-cassette; transforming the cell with a firstoligonucleotide comprising a nucleic acid sequence homologous to thefirst strand of a nucleic acid 5′ of the nucleic acid of interest; and asequence homologous to the first strand of a nucleic acid 3′ of thenucleic acid sequence of interest; and selecting for loss of the nucleicacid sequence encoding the reporter gene, wherein loss of the nucleicacid sequence encoding the reporter gene from the target double strandednucleic acid indicates deletion of the target double stranded nucleicacid.

[0191] Yet another provided embodiment is a method of deleting a targetdouble stranded nucleic acid sequence from within a cell, wherein thedouble-stranded nucleic acid cassette further comprises a nucleic acidsequence encoding a counterselectable marker located between the firstportion and the second portion, which cassette is referred to as aCORE-cassette, and wherein the method further comprises selecting forloss of both the nucleic acid sequence encoding the counterselectablemarker and the nucleic acid sequence encoding the reporter gene, whereinloss of both the nucleic acid sequence encoding the counterselectablemarker and the nucleic acid sequence encoding the reporter geneindicates deletion of the target double stranded nucleic acid.

[0192] Another embodiment is a method of deleting a target doublestranded nucleic acid sequence from within a cell, wherein thedouble-stranded nucleic acid cassette further comprises a nucleic acidsequence comprising a double-strand break recognition site, a nucleicacid encoding a double-strand break enzyme that recognizes thedouble-strand break recognition site, and an inducible promoter,operably connected with the nucleic acid encoding the double-strandbreak enzyme, which cassette is referred to as a RE-DSB-cassette.

[0193] In another provided method of deleting a target double strandednucleic acid sequence from within a cell, the double-stranded nucleicacid cassette further comprises a nucleic acid sequence comprising adouble-strand break recognition site, a nucleic acid encoding adouble-strand break enzyme that recognizes the double-strand breakrecognition site, and an inducible promoter, operably connected with thenucleic acid encoding the double-strand break enzyme, which cassette isreferred to as a CORE-DSB-cassette.

[0194] In specific embodiments that include in the cassette adouble-strand break site and nucleic acid sequence encoding adouble-strand break enzyme, the method further comprises inducingexpression of the double strand break enzyme, thereby stimulating adouble-strand break within the cassette, which double-strand breakstimulates recombination.

[0195] In specific examples of the provided methods of deleting a targetdouble stranded nucleic acid sequence, the first portion of the cassetteis homologous to a nucleic acid sequence on a first side of theinsertion point; and the second portion of the cassette is homologous toa second nucleic acid sequence on the second side of the insertionpoint.

[0196] Still other specific examples of such methods further involvetransforming the cell with a second oligonucleotide comprising a nucleicacid sequence homologous to the second strand of a nucleic acid 5′ ofthe target double stranded nucleic acid sequence; wherein the sequenceof the first oligonucleotide is homologous to at least 10 nucleotides atthe 3′ end of the sequence of the second oligonucleotide. In stillfurther specific examples, the sequence of the first oligonucleotide ishomologous to at least 15 nucleotides at the 3′ end of the sequence ofthe second oligonucleotide.

[0197] In yet other examples, the second oligonucleotide furthercomprises a sequence homologous to the second strand of a nucleic acid3′ of the nucleic acid sequence of interest.

[0198] In specific examples, the second oligonucleotide is fullycomplementary to the first oligonucleotide.

[0199] For specific examples of those embodiments that involve orrequire a counterselectable marker, the counterselectable marker isKlURA3, URA3, TRP5, TRP1, or a gene encoding a toxin. In particularexamples, the counterselectable marker is a gene encoding a toxin, andthe toxin is an inducible restriction enzyme or an inducible p53 gene.By way of example, the inducible p53 gene in some instances is a toxicversion.

[0200] In yet a further embodiment of a method of deletion of a targetdouble stranded nucleic acid, the reporter encodes a polypeptide thatconfers antibiotic resistance to the cell, for instance wherein theantibiotic is G418, hygromycin, kanamycin, ampicillin, tetracycline,chloramphenicol, neomycin, zeocin, nourseothricin, cycloheximide, orcanavanine. Alternatively, in other embodiments the reporter encodes apolypeptide from an amino acid or nucleotide synthesis pathway. Forinstance, the polypeptide is LEU2, TRP5, TRP1, LYS2, HIS3, or ADE2 inspecific examples.

[0201] In specific examples of methods for deleting a target doublestranded nucleic acid, the oligonucleotide is at least 30 nucleotides inlength., for instance at least 40, at least 50, or at least 80nucleotides in length. In other specific examples, the first and thesecond oligonucleotides are each at least 20 nucleotides in length, forinstance at least 30, at least 40, at least 50, or at least 80nucleotides in length.

[0202] In further embodiments, the first and the second oligonucleotideare of different lengths. In still other embodiments of these methods,the first and the second oligonucleotides are at least 20 nucleotides inlength and wherein the region of overlap at the 3′ ends is at least 10base pairs, or the first and the second oligonucleotides are at least 40nucleotides in length and wherein the region of overlap at the 3′ endsis at least 15 base pairs.

[0203] In particular example methods, the 3′ ends of the first andsecond oligonucleotide can be extended by in vitro polymerization.

[0204] In examples of the provided methods, the cell is a cell of anorganisms in which homologous recombination can be accomplished. By wayof example, the cell is a fungus cell (for instance, a yeast cell), abacteria cell, a plant cell, or an animal cell (for instance, a chickencell). Optionally, such cells can comprises a human chromosome orfragment thereof. Also provided in a further embodiment is a method forassessing a mutation in a nucleic acid sequence to determine if themutation affects a function or expression pattern of the nucleic acid,which method comprises a delitto perfetto mutagenesis method asdescribed herein. Another method is a method for analyzing a series of amutations in a nucleic acid sequence, which method involves performingthe method assessing a mutation in a nucleic acid sequence to determineif the mutation affects a function or expression pattern of the nucleicacid a plurality of times, wherein at least two different mutations areintroduced in the nucleic acid sequence; and analyzing the function orexpression pattern of the nucleic acid, thereby analyzing a series of amutations in the nucleic acid sequence.

[0205] In examples of these methods, the nucleic acid sequence is amammalian sequence. In other examples, the nucleic acid sequence encodesa polypeptide. In yet further examples, the method further comprisesassessing a function of the polypeptide.

[0206] Optionally, the described methods are carried out in a cell of ahaploid yeast, or in a cell of a diploid yeast.

[0207] Another embodiment is use of a described method as a diagnostictool wherein a series of strains or cell lines are created, each withthe cassette at a different position within a gene, such that mutationscan be introduced anywhere within a gene and the biological consequencesassessed.

[0208] Yet another embodiment is a method of analyzing defects in p53wherein a p53 mutant protein is expressed in yeast in such a way that animpact of a defect in the p53 mutant protein can be assessed.Optionally, such a method further comprises assessing a defect in thep53 mutant protein.

[0209] Yet further embodiments are CORE-cassette constructs,RE-DSB-cassette constructs, and CORE-DSB-cassette constructs for use indelitto perfetto mutagenesis.

[0210] Also provided in other embodiments are integrative recombinantoligonucleotides (IRO) for use in delitto perfetto mutagenesis.

[0211] Yet a further embodiment is a kit for carrying out in vivomutagenesis or deletion of a nucleic acid sequence, comprising an amountof a CORE-cassette construct , a RE-DSB-cassette construct, or aCORE-DSB-cassette construct, and an amount of an integrative recombinantoligonucleotide.

[0212] IV. Delitto Perfetto Mutagenesis

[0213] This disclosure provides mutagenesis systems based ontransformation with oligonucleotides, which systems provide for therapid creation of site-specific or random mutations in DNAs. In certainembodiments, the mutagenesis is carried out within the yeastSaccharomyces cerevisiae.

[0214] The two-step, cloning-free methods generate mutated products invivo having only the desired mutation, such as single or multiple basechanges, an insertion, or a small or a large deletion. Delitto perfettomutagenesis is extremely versatile. It enables multiple rounds ofspecific or random changes within a specified window of up to 200 bp,and in some embodiments even longer windows, for instance up to 220,250, 270, 290, 300, 350, 400 or more nucleotides.

[0215] The delitto perfetto process, which is at least partiallydependent on the RAD52 pathway, is not constrained by the distributionof naturally occurring restriction sites and requires minimal DNAsequencing. Since yeast is commonly used for random and selectivecloning of genomic DNA from higher eukaryotes such as YACs, thisstrategy also provides an efficient way to create precise changes withinmammalian DNA sequences.

[0216] The mutagenesis system is referred to herein generally as delittoperfetto (Italian: meaning perfect murder but used as an idiom forperfect deletion) because the introduction of the desired mutationinvolves the complete removal of a marker cassette (the CORE-cassette)previously integrated at the target locus.

[0217] As shown in FIG. 1, the first step is the introduction of aCORE-cassette containing a COunterselectable marker (e.g., KlURA3) and aREporter gene (e.g., kanMX4) near the site in which one or more changesare desired. The insertion of the CORE-cassette, using the highlyproficient homologous recombination system in yeast (Fincham, Microbiol.Rev. 53, 148-70, 1989 [published erratum appears in Microbiol. Rev.55(2):334, 1991]; Lewis and Resnick, Mutat. Res. 451:71-89, 2000; Sunget al., Mutat. Res. 451:257-275, 2000), is key to the delitto perfettomutagenesis approach.

[0218] Transformation of the cells that contain a CORE-cassette with oneor two Integrative Recombinant Oligonucleotides (IROs) leads to excisionof the CORE-cassette. Counterselection for loss of the counterselectablemarker, for instance the KlURA3 marker, followed by testing forsimultaneous loss of the reporter (e.g., the kanMX4 marker) providesselection for desired changes and minimizes false positives. The type ofmutation that results is dictated by the IRO(s) used in excising theCORE-cassette. Specific embodiments are described more fully below, andcertain embodiments are illustrated in the Examples.

[0219] While additional mutations may be associated with the varioustargeted nucleic acid changes using delitto perfetto mutagenesis, theyhave always been confined to the IRO regions (those regions of thetarget nucleic acid that share homology with the IRO(s)).

[0220] Site-Specific Point Mutagenesis

[0221] To generate site-specific point mutations using delitto perfettomutagenesis, one or both of the IROs used for transforming the cellscontains the desired mutation(s). One embodiment is illustrated in FIG.1, in the right hand pair of illustrated oligonucleotides. A single basemutation is indicated by the asterisk; in the illustrated embodiment,the mutation is found in both of the oligonucleotides (thus, is withinthe region of overlap or complementarity between the oligonucleotides).

[0222] In other embodiments, the point mutation is in one of theoverhanging regions of the oligonucleotide pair. See, for instance, theembodiment illustrated in FIG. 5C, which incorporates a singlenucleotide change into the target nucleic acid 40 nucleotides outside ofthe overlapping portion of the pair of oligonucleotides.

[0223] The further out a mutation is toward the end of an IRO, thehigher the odds that the transforming recombinational event will occur“inside” of the mutation, which yields a cell that has lost theCORE-cassette without incorporating the desired point mutation. Incertain embodiments the point mutation are no closer than about 20nucleotides, for instance no closer than about 40 nucleotides, to theend of an IRO. This position will allow sufficient nucleic acid sequencein which the homologous recombination can occur, without excluding thepoint mutation.

[0224] It is also possible to incorporate more than one point mutationinto a target nucleic acid sequence simultaneously, by using IRO(s) thatcontain more than one nucleic acid change compared to the target. Incertain embodiments all of these multiple mutations are no closer thanabout 20 nucleotides, for instance no closer than about 40 nucleotides,from the end of an IRO.

[0225] Random Mutagenesis

[0226] As illustrated schematically in FIG. 5B, the delitto perfettoapproach can also be used for random mutagenesis directly in the genome.Oligonucleotides with degenerate (i.e., random) sequence arecommercially available, for instance from Invitrogen or other suppliersof oligonucleotides. The region of the IROs that is not in the overlapcan be designed for random mutagenesis, simply by generating (e.g.,ordering from a supplier) a random oligonucleotide, including a sequencewith one or more ambiguous bases (example: N=A+C+T+G, R=A+G, etc.). Thisapproach can be used to concurrently generate several or many differentmutants in a defined DNA region.

[0227] By way of example, random mutagenesis methods can be applied, forinstance, to structure-function studies, particularly for targetingmultiple and mutations to key regions within a sequence such as specificprotein motifs (which are usually short), or to defined loops (which aretypically around 10 to 20 residues in length).

[0228] Deletion Mutagenesis

[0229] The delitto perfetto mutagenesis system also can be used todelete sequences from within a nucleic acid molecule. Such deletions canbe short (i.e., a few to several nucleotides, such as a portion of acoding sequence or regulatory sequence) or longer (i.e., an entire geneor region of a chromosome). Deletion of a relatively long nucleic acidsequence is schematically illustrated in FIG. 1, with the left-hand pairof oligonucleotides. In the illustrated embodiment, the IROs have beendesigned to contain nucleic acid sequence that is both upstream anddownstream of the inserted CORE-cassette (indicated with hatched andbroad slashed boxes on the gene sequence and primers). When the IROsmediate homologous recombination and loss of the CORE-cassette, therecombination events take place well outside of the CORE-cassette, andthus the entire region of nucleic acid from within the hatched area towithin the broad slashed area is lost.

[0230] In other embodiments, one of the IROs (or both of them) willcarry a short deletion mutation, for instance of one to 15 nucleicacids. This enables the introduction of short deletions, using amechanism essentially similar to that observed for the site-directedmutagenesis embodiments described above.

[0231] Deletion Mutagenesis of Essential Genes

[0232] Similarly, delitto perfetto mutagenesis can be used to generatedeletions of genes that are essential to the cell in which themutagenesis is being carried out. In this embodiment, a copy of theessential gene is transformed into the cell on, for instance, a plasmid.This plasmid copy of the gene provides function (in some examples, underinducible or inhibitable control) while the other (e.g., genomic) copyof the gene can be mutated at will using delitto perfetto mutagenesis.An example of the mutagenesis of essential genes is provided below.

[0233] General Mutagenesis of Essential Genes

[0234] Mutations can also be created in essential genes using delittoperfetto mutagenesis, for instance by inserting the CORE cassettedownstream from the stop codon of the target essential gene and usingoligonucleotides or larger PCR products to create non-lethal mutations.

[0235] Delitto perfetto Mutagenesis on Genes Encoded on YACs

[0236] Sequencing of the genomes of several higher eukaryotes, includinghumans, has produced a need for functional evaluation of large numbersof genes. However, the feasibility of characterizing large, complex DNAmolecules is limited by the difficulty in generating appropriatespecific mutations. A disadvantage of present systems for modificationof large DNA molecules is that they all require in vitro mutagenesis,which depends on laborious subcloning of the region to be mutagenized.See, for instance, Barton et al., Nucleic Acids Res. 18, 7349-7355,1990; McCormick et al., Proc. Natl. Acad. Sci. USA 92:10147-10151, 1995;Peterson et al., Trends Genet. 13:61-66, 1997; Boren et al., Genome Res.6:1123-1130, 1996; Callow et al., Nucleic Acids Res. 22:4348-4349, 1994;Tucker and Burke, Nucleic Acids Res. 24:3467-3468, 1996; and Nefedov etal., Nucleic Acids Res. 28:E79, 2000.

[0237] The disclosed mutagenesis methods expand opportunities forfunctional analysis of mammalian DNAs, or any nucleic acid sequence(e.g., a genomic sequence) cloned on a YAC. Yeast has proven ideal forthe isolation and propagation of specific chromosomal regions and genesfrom higher eukaryotes. For example, TAR (transformation-associatedrecombination) cloning (Larionov et al., Proc. Natl. Acad. Sci. USA93:491-496, 1996) can be used for the isolation of functional humangenes, such as BRCA 1, BRCA2 and HPRT (Larionov et al., Proc. Natl. AcadSci. USA 94, 7384-7387, 1997; Kouprina et al., Proc. Natl. Acad Sci. USA95:4469-4474, 1998). The delitto perfetto methods provide a convenienttool for modifying TAR-cloned genes.

[0238] Delitto perfetto to Characterize Protein Domains

[0239] The delitto perfetto mutagenesis approach can be applied tocharacterize specific motifs or domains of proteins. This may beparticularly interesting for studies of genes associated with humandiseases. The yeast S. cerevisiae is one of the most used model systemsto study and analyze function of expressed heterologous genes. Severalassays have been established in yeast to characterize protein functionand analyze the effect of defined protein modifications, includingADE2-color assay for p53 mutants (Flaman et al., Proc. Natl. Acad Sci.92: 3963-3967, 1995), growth assay for p53 mutants (Inga et al.,Oncogene 20:501-5 13, 2001), growth assay for human FEN-1 mutants(Greene et al, Hum. Mol. Genet. 8: 2263-2273, 1999); anhsRPB4/7-dependent yeast assay for trans-activation by the EWS oncogene(Zhou and Lee, Oncogene 20:1519-1524, 2001); a cell-based screen inyeast for the identification of novel inhibitors of poly(adp-ribose)polymerase/parp1 and parp2 (Perkins et al, Cancer Res. 61: 4175-4183,2001); yeast-based transcription assay for functional analysis of humanBRCA1 C-terminal missense mutations identified in breast and ovariancancer families (Vallon-Christersson et al, Hum. Mol. Genet. 10:353-360, 2001).

[0240] Adapted Yeast Backgrounds for Delitto perfetto Mutagenesis

[0241] Specific mutant yeast strains such as those carrying mutations inor that disrupts a recombination system can be used to enhance targetingof genes in the delitto perfetto mutagenesis system. Specific examplesinclude hpr1, hpr5 and rsc1 mutants in yeast; see, for instance,Aguilera and Klein, Genetics 119:779-790, 1988; Aguilera and Klein, Mol.Cell. Biol. 10:1439-1459, 1990; Schneiter et al., Mol. Cell. Biol.19:3415-3422, 1999; Gallardo and Aguilera, Genetics 157:79-89, 2001;Cairns et al., Mol. Cell 4:715-723, 1999; and Goodwin and Nicolas, Gene268:1-7, 2001. In particular, these mutants are thought to lead toincreased spontaneous recombination as well as enhancing targeting ofthe CORE-cassette. These mutants along with mutations in genes affectingnuclease (e.g., the exo1 or mre11 nucleases; Tiskoff et al., Proc. Natl.Acad. Sci. USA 94:7487-7492, 1997; Tran et al., Mol. Cell. Biol.19:2000-2007, 1999; Bressan et al., Mol. Cell. Biol. 19:7681-7687, 1999;and Symington et al., Nuc. Acids Res. 28:4649-4659, 2000), including thespecific mre11 D16A mutant, are also expected to lead to increasedefficiency of IRO targeting. For example, IROs are expected to be morestable in the nuclease mutants.

[0242] Although the listed examples are in the yeast system, similarexamples of mutations that are advantageous for delitto perfettomutagenesis are found in other organisms, for instance mutants in therecBC gene of E. coli.

[0243] Mutagenesis in Diploid Yeast

[0244] The delitto perfetto system can be applied to diploid yeaststrains as well as haploids. For example, the CORE-cassette is insertedinto one of the pair of homologues in a diploid strain, essentially asexplained for insertion in a haploid strain. Transformation of thatstrain with one or a pair of IROs leads to loss of CORE-cassette andcorresponding replacement within the genome. Due to recombination duringcell division, prior to transformation with the IRO(s) there may be asubstantial background of homozygous CORE-cassette deletions. However,the efficiency of targeting is sufficiently high (comparable tohaploids) to make the delitto perfetto strategy worthwhile in diploids.Furthermore, use of targeting mutants described above, especially thenuclease mutants, is expected to increase the relative frequency ofdelitto perfetto events in comparison to the background of CORE-cassetteminus cells.

[0245] Delitto Perfetto in Other Species

[0246] Although several of the illustrative embodiments are disclosed ina yeast (S. cerevisiae) system, it is believed that the delitto perfettomutagenesis methods work in all biological organisms that have afunctional recombination system, even where the recombination system isnot as proficient as in yeast. Other cells or cell types that have afunctional homologous recombination systems include bacteria such asBacillus subtilis and E. coli (which is RecE RecT recombinationproficient; Muyrers et al., EMBO rep. 1: 239-243, 2000); protozoa (e.g.,Plasmodium, Toxoplasma); other yeast (e.g., Schizosaccharomyces pombe);filamentous fungi (e.g., Ashbya gossypii); plants, for instance the mossPhyscomitrella patens (Schaefer and Zryd, Plant J. 11: 1195-1206, 1997);and animal cells, such as mammalian cells and chicken DT40 cells (Diekenet al., Nat. Genet. 12:174-182, 1996). Application to DT40 cells isespecially useful since cell lines with specific human chromosomes areavailable (Koi et al., Cytogenet. Cell Genet. 76:72-76, 1997). It isalso possible to use mammalian cells, since some lines exhibit higherrates of recombination (see, e.g., Bunz et al., Science 282: 1497-1501,1998).

[0247] Plasmids used in yeast can be easily shuttled to E. coli.Alternatively, the system is believed to apply to a variety of otherorganisms capable of recombination, such as E. coli and chicken cells,which in each case may be engineered to contain human chromosomes orportions thereof.

[0248] Production of Oligonucleotides

[0249] In vitro methods for the synthesis of oligonucleotides are wellknown to those of ordinary skill in the art; such conventional methodscan be used to produce IROs for the disclosed methods. The most commonmethod for in vitro oligonucleotide synthesis is the phosphoramiditemethod, formulated by Letsinger and further developed by Caruthers(Caruthers et al., Chemical synthesis of deoxyoligonucleotides, inMethods Enzymol. 154:287-313, 1987). This is a non-aqueous, solid phasereaction carried out in a stepwise manner, wherein a single nucleotide(or modified nucleotide) is added to a growing oligonucleotide. Theindividual nucleotides are added in the form of reactive3′-phosphoramidite derivatives. See also, Gait (Ed.), OligonucleotideSynthesis. A practical approach, IRL Press, 1984.

[0250] In general, the synthesis reactions proceed as follows: First, adimethoxytrityl or equivalent protecting group at the 5′ end of thegrowing oligonucleotide chain is removed by acid treatment. (The growingchain is anchored by its 3′ end to a solid support such as a siliconbead.) The newly liberated 5′ end of the oligonucleotide chain iscoupled to the 3′-phosphoramidite derivative of the next deoxynucleosideto be added to the chain, using the coupling agent tetrazole. Thecoupling reaction usually proceeds at an efficiency of approximately99%; any remaining unreacted 5′ ends are capped by acetylation so as toblock extension in subsequent couplings. Finally, the phosphite triestergroup produced by the coupling step is oxidized to the phosphotriester,yielding a chain that has been lengthened by one nucleotide residue.This process is repeated, adding one residue per cycle. See, forinstances, U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 4,973,679,and 5,132,418. Oligonucleotide synthesizers that employ this or similarmethods are available commercially (e.g., the PolyPlex oligonucleotidesynthesizer from Gene Machines, San Carlos, Calif.). In addition, manycompanies will perform such synthesis (e.g., Sigma-Genosys, Tex.; OperonTechnologies, Calif.; Integrated DNA Technologies, Iowa; and TriLinkBioTechnologies, Calif.).

[0251] Modified nucleotides can be incorporated into an oligonucleotideessentially as described above for non-modified nucleotides.

[0252] Random primers may be generated using known chemical synthesisprocedures; randomness of the sequence may be introduced by providing amixture of nucleic acid residues in the reaction mixture at one or moreaddition steps (to produce a mixture of oligonucleotides with randomsequence). See, for instance, U.S. Pat. Nos. 5,043,272 and 5,106,727. Arandom primer preparation (which is a mixture of differentoligonucleotides, each of determinate sequence) can be generated bysequentially incorporating nucleic acid residues from a mixture of, forinstance, 25% of each of dATP, dCTP, dGTP, and dTTP, (or a modified dNTPsuch as aa-dUTP). Other ratios of dNTPs can be used (e.g., more or lessof any one dNTP, with the other proportions adapted so the whole amountis 100%). Likewise, in the synthesis of a random primer, the synthesizercan be programmed to introduce one or more known residues (such as oneor more specific nucleotide residues or modified nucleotide residues) ata defined location within the primer. For instance, a defined sequencecan be in the middle of the primer (see FIG. 5B) that is used alone orcoupled with a second primer (with random sequences to the 5′ and 3′end), or a combination of these.

[0253] Presently, oligonucleotides are conveniently availablecommercially up to approximately 125 nucleotides; beyond this length theefficiency and purification drops. As the available size ofoligonucleotide amenable to in vitro synthesis is increased, theapplicability of delitto perfetto mutagenesis will be increased in termsof size of window available for mutational modification.

[0254] Applications of Delitto Perfetto Mutagenesis Techniques

[0255] Additional applications of delitto perfetto mutagenesis includedtargeted changes in the genome of various organisms, including yeast;modification of large human genes, for instance cloned in yeast as YACs,which could readily be moved to other species (e.g., E. coli or humancells) after mutagenesis; and larger windows of site-directedmutagenesis as the synthesis length of oligonucleotides becomes moreaccessible.

[0256] V. Delitto Perfetto-DSB Mutagenesis

[0257] Embodiments described above provide methods for targetedmodification of the Saccharomyces cerevisiae genome, for instance,through homologous recombination and oligonucleotides. The ability toengineer specific changes directly into the genome so that no trace ofthe targeting material remains is generally a laborious, multistepprocess with limitations on the types of alterations. Furthermore,site-directed mutagenesis and functional analysis of changes can belabor intensive and low efficiency. The delitto perfetto processdescribed above allows for the generation of products having only thedesired mutation, such as a single or multiple base change, aninsertion, a small or a large deletion. There is a clear need for ageneral usage of this technique that would allow for a) independencefrom the genetic background of a strain, b) possibility to accomplishlarge genome rearrangements, c) modifications in essential genes, evenin genes that affect homologous recombination and d) the possibility ofgreater efficiency.

[0258] The delitto perfetto-DSB system is another embodiment of thedelitto perfetto process, which provides a dramatic increase inefficiency, with an over 1000-fold increase in mutation efficiency. Thisresults in considerably greater versatility and high throughputgeneration of genetic alterations using this embodiment. This novelsystem originates form the combination of the following factors: thegeneration of an unique double-strand break (DSB) in the genome tostimulate targeting, the use of an endonuclease enzyme that generates aDSB at a unique short DNA site, and the possibility to turn on and offthe endonuclease enzyme using an inducible promoter.

[0259] In addition to its relevance as a fundamental biological process,DSB-mediated recombination is the basis of gene modification in yeastand in other eukaryotes (reviewed in Paques and Haber, Microbiology andMolecular Biology Reviews 1999, 63:349-404). The repair of DSBs in S.cerevisiae seems to occur predominantly by recombination between thebroken ends of a DNA molecule and intact homologous sequences. DSBsgreatly stimulate homologous recombination: DSBs are the soleinstigators of recombination in meiotic cells and are a major factor inrecombination in mitotic cells. DSBs can appear as a consequence ofionizing radiation, by mechanical stress, by replication of asingle-stranded nicked chromosome, or by endonucleases expressed invivo.

[0260] In mitotic cells, initiation of recombination can be accomplishedby the induction of a site-specific specific endonuclease. Two suchsystem have been developed in yeast. The HO endonuclease recognizes adegenerate target sequence of 22 bp, and normally cleaves only one sitein the entire yeast genome, the mating-type (MAT) locus. Constructs inwhich the HO gene is fused to a galactose-inducible promoter have madeit possible to express HO simply by adding galactose.

[0261] A second endonuclease is I-SceI, normally encoded and expressedonly in yeast mitochondria to facilitate the movement of a mobile intron(cut site produced below). A synthetic version of this gene, replacingcodons whose usage is different in the mitochondria and the cytoplasm,was constructed, again under the control of a galaxies-induciblepromoter (Plessis et al., Genetics 130:451-460, 1992). A 45- to90-minute induction of either HO or I-SceI leads to the cleavage of asignificant fraction of target sites.

[0262] I-SceI site               ↓ 5′ - TAGGGATAACAGGGTAAT- 3′ 3′ -ATCCCTATTGTCCCATTA - 5′           ↑

[0263] Use of a CORE-DSB Cassette for Delitto Perfetto-DSB Mutagenesis

[0264] This embodiment provides certain advantages over the delittoperfetto embodiment, with an over 1000-fold increase in efficiency.Thus, the delitto perfetto-DSB embodiments provide greater versatilityand permit higher throughput generation of genetic alterations. Thisembodiment is based on the introduction of a single DSB within aCORE-cassette in the targeted region at the time of transformation usingan in vivo, tightly regulatable system (see FIG. 7). Through the use ofthis novel system we have established that oligonucleotide modificationscan be created in up to 5% of all cells, an extremely highly efficientprocess. Since the system provides for the generation of multiplemutations in 200 bp or larger windows, it is useful in the modificationof genes and proteins that are of health relevance, and for thegeneration of diversity within proteins.

[0265] The delitto perfetto-DSB system greatly enhances opportunitiesfor the rapid modification of genes and DNAs, diversification of genes,alteration of genes in diploid cells, the use of targeting cassettesthat do not depend on counterselection (e.g., RE-DSB, as describedbelow), in vivo modification of essential genes, manipulation of largechromosomal regions, and gene modification in cells deficient inrecombination.

[0266] The utility of the system extends to all the features of thedelitto perfetto system including: it does not require subcloning, itdoes not rely on the need for unique restriction sites and it does notrequire resequencing of large regions of DNA once a modification ismade. Only the region corresponding to the oligonucleotides need besequenced. The system could be used in other organisms whererecombination is proficient, for example in chicken DT40 cells which areused for examining human chromosomes, or in cells such as human whereDSBs can stimulate recombination. This could have a variety of healthbenefits, especially in the areas of gene therapy and proteindevelopment.

[0267] Shortened CORE-DSB-cassettes (Without Counterselection); RE-DSBMutagenesis

[0268] In another embodiment, due to the high-efficiency mutagenesisthat results from using the DSB system, the counterselectable marker canbe omitted from the cassette.

[0269] Since the mutagenesis approach that utilizes the CORE-DSB ishighly efficient, between 2 to 5%, it is possible to isolate the clonescontaining the desired change in the genome directly on a nonselectivemedia (YPD plate), without the use of the counterselectable marker. Inthis case, the delitto perfetto-DSB approach can be accomplished with aCORE-DSB cassette that lacks the counterselectable marker, referred toas RE-DSB (for example GAL1/10:: I-SceI+kanMX4 or +HygroR).

[0270] Externally Added DSB Enzyme

[0271] In another embodiment, the CORE-DSB-cassette does not contain asequence encoding a double-strand break enzyme, and instead thedouble-strand break function is provided by adding exogenousdouble-strand break enzyme to the cells after the CORE-DSB-cassette (orRE-DSB-cassette) has been integrated into the target sequence. Theapproach of adding an external enzyme to cut at a unique site in vivo toinduce recombination has been used with various mammalian cell systems,including the use of I-SecI. See, for instance, Lin et al., (Mol. CellBiol. 19:8353-60, 1999) and Johnson and Jasin (Biochem. Soc. Trans.29:196-201, 2001).

[0272] Generation of Mutations in Diploid Organisms

[0273] The delitto perfetto-DSB approach described herein providesenhanced opportunities to accomplish mutagenesis in diploid yeast.Normally, mutations in diploid organisms are often masked because theyare recessive and therefore undetectable. The delitto perfetto-DSBsystem provides for recognition of a mutational change by virtue of lossof the cassette event and therefore enables the identification andisolation of diploid clones with the desired change. While loss ofheterozygosity may also occur, the frequency of the targeted changes aresufficiently large that they can be identified in a relatively smallnumber of clones, such as 1 in 50, 1 in 100, or 1 in 200.

[0274] Generation of Diversity Within Proteins

[0275] The delitto perfetto-DSB system enables very efficient targetingmediated by oligonucleotides. The possibility to obtain a huge number oftargeted clones can be used, for instance, to create multiple variantsof a protein product of a gene of interest. This diversity of proteinproducts can be obtained if the gene of interest is integrated into theyeast genome (or into another genome where homologous recombination isalso proficient), or is cloned on a plasmid. The insertion of theCORE-DSB or RE-DSB cassette within the gene at the desired locus,followed by induction of the DSB, allows for rapid creation of manyvariants, upon transformation with IROs designed for random mutagenesis.This provides the possibility of at least one and up to many changes perIRO-targeted event, thereby providing opportunities for diversifyinggenes (or protein products).

[0276] This approach to random modification of genes directly in vivousing IROs is expected to be much more efficient than in vitroapproaches to random mutagenesis using oligonucleotides. If the gene iscloned on a multicopy vector (such as a yeast 2μvector) the number ofvariant forms of the gene generated can be up to 1000-fold, forinstance, 100-fold, 250-fold, or 500-fold, higher (as described below)than if a single copy vector or a genomic locus is used. For instance,it is believed that up to 10⁸ different variants can be obtained from asingle transformation event. In some embodiments, the random mutagenesisapproach is performed to isolate a particular variant form of thederived protein that gives rise to a selectable phenotype.

[0277] Generation of Intracellular Plasmid Variants by OligonucleotideGap Repair of an in vivo Linearized Multicopy Vector

[0278] The cassette CORE-DSB or RE-DSB can be cloned in vitro at adesired site within a desired gene on a multi copy vector (for instance,YEps (yeast episomal plasmids), 2-micron (2μ) derivative vectors foryeast Saccharomyces cerevisiae). The YEp vector containing the CORE-DSBor the RE-DSB is transformed by standard procedure into yeast cells.Inside the yeast cells the copy number of each YEp plasmid isspontaneously increased to 50-100 or more copies per cell.

[0279] Immediately prior to transformation with IROs cells are grown inthe presence of the inducer (i.e., galactose), which induces thesite-specific endonuclease expression (i.e. I-SceI). The enzyme targetsits single site in the cassette on each YEp vector and generates a DSB,linearizing all vectors in each cell. IROs are designed for randommutagenesis, as described herein.

[0280] Cells are transformed with IROs and selected for the presence ofthe plasmid. Transformation of cells with IROs leads to circularizationof the vectors, loss of the cassette and creation of the random mutation(theoretically, a different mutation is created in each vector). Sinceeach cell contains several vectors, each cell should contain manydifferent variants of the same gene.

[0281] This approach could be useful to increase the number of genevariants obtained by random mutagenesis of additional 10- to 100-fold ormore.

[0282] Increased Efficiency of PCR Targeting, and Modification ofEssential Genes

[0283] The delitto perfetto-DSB mutagenesis approach using the CORE-DSBor the RE-DSB utilizes IROs for making genome modification. However, thesame approach can also be applied using a PCR product (in place ofIROs), previously modified by random mutagenesis or site-directedmutagenesis in vitro or in vivo. The efficiency of PCR targetingdirectly in vivo in the genome is also increased about 1000-fold afterDSB induction.

[0284] This can be applied to all nonessential genes and even toessential genes directly if the cassette is inserted immediately afterthe stop codon of the gene.

[0285] For instance, a CORE-DSB or RE-DSB cassette can be insertedimmediately after the stop codon of a chosen essential gene, withoutdisrupting the gene. After induction of the DSB, a modified PCR productof the chosen gene can replace the cassette and the wild copy of thegene, leaving in the same locus a modified version of the gene. IROs canalso be used but in this case only the 3′ part of the gene (the partclose to the cassette) can be modified.

[0286] Co-Transformation of Oligonucleotides and a Vector

[0287] The extremely high efficient transformation produced with delittoperfetto-DSB and oligonucleotides allows the possibility to accomplishco-transformation experiments efficiently: transformation of IROs and aplasmid in one step. This allows for coincident modification of a genealong with the specific expression of another gene. For example, a geneon a plasmid might be lethal and another gene might impact on thelethality such that deletion of the impact gene enables survival..Therefore, could create a set of mutations in the vicinity of a CORE-DSBsite in the impacting gene such that the mutants enable survival withthe lethal gene. Also, it may be possible to include the gene for theDSB cutting enzyme on a plasmid so that the cuts are produced at thetime of IRO plus plasmid transformation.

[0288] DSB-based Mutagenesis in Low-recombination Strains and Cells

[0289] As discussed herein, in the delitto perfetto system, theintegration of IROs (whether presented singly or as pairs to cells)replacing the original CORE-cassette, was dependent on the RAD52 geneand appeared to be partially dependent on RAD50and MREll. It has nowbeen surprisingly found that, once a CORE-DSB (or RE-DSB) cassette isplaced into the genome, the DSB-enhanced approach can be used even inthe absence of a highly efficient recombination system.

[0290] By way of example, one could target to a recombination gene,i.e., inactivate the recombination gene and create mutations in it.Since the induction of a DSB in the integrated cassette results inextremely efficient targeting using IROs, the change in targetingefficiency is detectable even in the absence of a highly efficientrecombination system, for instance in a genetic situation with aRAD52-null gene (see, e.g., Example 8).

[0291] Genome-level Rearrangements

[0292] Use of delitto perfetto mutagenesis (with or without DSB) permitsgeneration of wide genome rearrangements. By way of example, suchrearrangements included large-scale deletions, such as thosedemonstrated herein for up to 16 kb using a delitto perfettoCORE-cassette. Since the use of the CORE-DSB or the RE-DSB provides forabout 1000-fold higher efficiency of targeting, large deletions such asthese, and larger, can be accomplished with a very high efficiency.Substantially larger deletions (e.g., up to 100 kb) are contemplated andexpected to be produced efficiently using CORE-DSB or RE-DSB-basedmutagenesis. This may find applications in the modifications of YACclones, for example.

[0293] Other kinds of contemplated rearrangements include chromosomecircularization. Previously this was accomplished by linearized plasmidtransformation with regions homologous to the ends of a chromosome, ahighly inefficient process (Bennett et al., Mol. Cell. Biology, 12:5359-5373, 2001). A cassette can be integrated near to a telomericregion of a chosen chromosome. Then IROs (single or a pair) can bedesigned containing at the 5′ end a sequence homologous to thedownstream region of the left telomere and at the 3′ end a sequencehomologous to the upstream region of the right telomere. Addition of theIRO(s) to the cell will cause the elimination of the cassette and ofboth the telomeres; therefore, the chromosome will be circularized.

[0294] The invention is illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Site-directed Delitto Perfetto Mutagenesis

[0295] The delitto perfetto approach permits genomic modificationwithout the retention of heterologous sequence (FIG. 1). Sinceintegration of the CORE-cassette utilizes simple, straightforwardintegrative transformation procedures (Wach et al., Yeast 10, 1793-1808,1994) and the mutation replacement involves transformation by easilydesigned oligonucleotides, this system provides for the rapid creationof a variety of genetic changes, as discussed below. Unlike othersystems, this in vivo site-specific mutagenesis strategy is versatilewith regard to chromosomal change, since many types of modification canbe generated in the nearby region once a CORE-cassette is inserted.

[0296] This example provides a description of several sequencemodifications that have been made using the delitto perfetto in vivomutagenesis system.

Experimental Protocols

[0297] Unless otherwise stated, genetic manipulations were carried outaccording to standard methods (Sambrook et al., (eds.), MolecularCloning: A Laboratory Manual, CSH Laboratory Press, Cold Spring Harbor,N.Y., 1989).

[0298] Yeast strains and growth conditions. Yeast strains used were:BY4742 (MATα, his3Δ1, leu2 Δ 0, lys2 Δ 0, ura3 Δ 0; Brachmann et al.,Yeast 14:115-132, 1998), VL6α (MATα, met14,lys2-801, his3-Δ 200, trp1-Δ63, ura3-52, ade2-101; Lewis et al., Mol. Cell Biol. 18:1891-1902, 1998)and E133 (S1-A12: MATα, ade5-1, lys2-12A, trp1-289, his7-2, leu2-3, 112,ura3-52; Tran et al.,Mol. Cell Biol. 17:2859-2865, 1997).

[0299] Cells were grown in standard rich (YPD), glycerol (YPG) andsynthetic minimal medium without uracil (SD Ura⁻), leucine (SD Leu⁻), ortryptophan (SD Trp⁻) (Sherman et al. (eds.) Methods in Yeast Genetics,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).Geneticin (G418) resistant cells were grown on YPD plates containing 200μg/ml of G418 (Gibco BRL, Grand Island, N.Y.) (Wach et al., Yeast 10,1793-1808, 1994). Ura⁻ cells were selected on synthetic complete mediumcontaining 5-fluoroorotic acid (5-FOA) (Toronto Research Chemicals Inc.,North York, Ontario, Canada) at 1 mg/ml (Boeke et al., Mol. Gen. Genet.197:345-346, 1984).

[0300] Plasmids. The plasmid pCORE was constructed by cloning a 1.5 kbBamHI-HincII fragment containing the kanMX4 gene (Wach et al., Yeast 10,1793-1808, 1994) into the BamHI-SspI sites of pFA6aKlURA3 (Delneri etal., Yeast 15:1681-1689, 1999).

[0301] Plasmid pBL230 (Ayyagari et al., Mol. Cell Biol. 15:4420-4429,1995) harbors genomic DNA containing the POL30 gene (from 195 bpupstream of the ATG to 170 bp downstream of its stop codon).

[0302] Deletion of RAD52 was made using plasmid pΔ52Leu (Lewis et al.,Mol. Cell Biol. 18:1891 -1902, 1998) cut with ApaI and NotI.

[0303] pLKL67Y was created by cloning a 1.2 kb URA3 fragment frompRS315+URA3 into the XhoI-SacI sites of the multiple-restriction site(of the centromeric vector pRS313 (Sikorski and Hieter, Genetics122:19-27, 1989).

[0304] Yeast Transformation. Transformation of yeast with circularplasmids, linear double-stranded stranded DNA molecules, oroligonucleotides was performed according to a high efficiency lithiumacetate protocol as previously described (Gietz, “High efficiencytransformation with lithium acetate” in: In Molecular Genetics of Yeast.A Practical Approach. (ed. Johnston) IRL Press, Oxford, UK, 121-134,1994).

[0305] PCR Amplification and Sequence Analysis. The kanMX4 and KlURA3CORE-cassette was amplified as a 3.2 kb DNA fragment from pCORE usingTaq DNA polymerase (Roche, Indianapolis, Ind.), with 32 cycles of 30seconds at 94° C., 30 seconds at 56° C., and 3 minutes 20 seconds at 72°C. For integration of the CORE-cassette into chromosomal loci, chimeric70-mers were designed, consisting of 50 nucleotides homologous to theappropriate flanking region of the genomic target locus and 20nucleotides, shown here, which allow for the amplification of theCORE-cassette:

[0306] 5′- . . . GAGCTCGTTTTCGACACTGG-3′ for the kanMX4 side (SEQ IDNO: 1) and

[0307] 5′- . . . TCCTTACCATTAAGTTGATC-3′ for the KlURA3 side (SEQ ID NO:2).

[0308] Amplification using these 70-mers yielded PCR product with 50nucleotides each of the upstream and downstream chromosomal sequence,flanking the CORE-cassette itself.

[0309] To identify clones with correct CORE-cassette integration, colonyPCR (Huxley et al., Trends Genet. 6:236, 1990) was performed usingprimers designed for annealing upstream and downstream of theintegration locus and within the CORE-cassette.

[0310] PCR fragments (about 0.5 kb) for the new mutant constructs,together with their corresponding wild-type loci, were purified byphenol-chloroform extraction, purified using the QIAquick PCRPurification Kit (Qiagen USA, Valencia, Calif.) and used for DNAsequencing. Sequencing reactions were performed on both strands usingthe dRhodamine Terminator Cycle Sequencing Kit (ABI Prism, AppliedBiosystems, Foster City, Calif.) and run in an automatic sequencingmachine (AB1377; Applied Biosystems).

[0311] Initial insertion of the CORE cassette into genes MLP2, POL30,TRP5 and SIR2 (within codon 270 and between codons 354 and 355) wasperformed using the following primers, respectively:MLP2.U/MLP2.G,PCNA.U/PCNA.G, TRP5.U/TRP5.G, SIR2.U1 /SIR2.G1, and SIR2.U2/SIR2.G2. K2and URA3.2 are internal primers in the CORE cassette, within kanMX4 andKlURA3, respectively.

[0312] CORE insertion was verified at each locus using the followingprimer combinations: MLP2.1/MLP2.2/K2 and MLP2.3/MLP2.4/URA3.2;PCNA.3/PCNA.4, PCNA.3/URA3.2 and PCNA.4/K2; TRP5.1/TRP5.2, TRP5.1/K2 andTRP5.2/URA3.2; SIR2.1/SIR2.4/K2 and SIR2.1/SIR2.4/URA3.2 for COREinsertion in codon 270 of SIR2; SIR2.2/SIR2.3/K2 andSIR2.2/SIR2.3/URA3.2 for CORE insertion between codons 354 and 355 ofSIR2.

[0313] CORE-cassette excisions and sequence analyses were carried outusing the following primers: MLP2.1/MLP2.4; PCNA.3/PCNA.4;TRP5.1/TRP5.2; SIR2.1/SIR2.4 for CORE excision from codon 270 of SIR2and SIR2.3/SIR2.2 for CORE excision between codons 345 and 364 of SIR2.

[0314] Primers used to verify deletion of RAD52: RAD52.1/LEU2.2 andRAD52.4/LEU2.1.

[0315] Integrative Recombinant Oligonucleotide (IRO) Mutagenesis

[0316] IROs used in this study differ by size and orientation withrespect to the CORE-cassette integration point.

[0317] Oligonucleotides were used in transformation experiments assingle species, or in two-species combinations as shown in FIG. 3. Toextend the sequence of the 3′-overlapping IROs (a+b) in vitro, a 50-μlreaction mix contained 0.5 nmoles of each primer, 4 units Platinum Pfx(Gibco BRL), 5 μl 10× Buffer, 1 μl 50 mM MgSO4 and 2 μl 10 mM dNTPs(Roche). Extension was performed as follows: 1 minute at 94° C. and 30seconds to 3 minutes at 68° C. Samples were ethanol precipitated andresuspended in 10-20 μl water. The use of Taq polymerase (Roche) insteadof Platinum Pfx DNA polymerase for these extension reactions partiallyreduced the efficiency of oligonucleotide integration.

[0318] Before adding IRO DNA to the cells, the single oligonucleotidespecies or the two oligonucleotide species-combinations, except for thepair extended in vitro with Pfx DNA polymerase (a+b Pfx), were denaturedat 100° C. for 2 minutes and then placed immediately on ice in order toreduce or eliminate possible secondary structure.

[0319] IROs used in this Example are described with numbers indicatingthe DNA sequences spanned by each oligonucleotide, starting from thefirst 5′ nucleotide and terminating with the last 3′ base relative tothe CORE-cassette integration point shown in FIG. 2.

[0320] The IROs used to delete MLP2 were MLP2.a (5′−70 . . . −1, +1 . .. +10) and MLP2.b (5′+70 . . . +1, −1 . . . −10).

[0321] IROs used to generate the POL30 deletion were PCNA.a (5′−848 . .. −778, +1 . . . +10) and PCNA.b (5′+70 . . . +1, −778 . . . −788).

[0322] IROs used to generate the silent mutation in the codon 334 ofTRP5 were TRP5.a (5′−70 . . . −1, +1 . . . +10), TRP5.b (5′+70 . . . +1,−1 . . . −10), TRP5.c (5′+10 . . . +1, −1 . . . −70), TRP5.d (5′−10 . .. −1, +1 . . . +70), TRP5.e (5′−47 . . . −1, −1 . . . +48), TRP5.f(5′−48 . . . +1, −1 . . . −70), TRP5.d (5′−40 . . . −1, +1 . . . +41),TRP5.f1 (5+41 . . . +1, −1 . . . −40), TRP5.e2 (5′−25 . . . −1, +1 . . .+26), TRP5.f2 (5′+26 . . . +1, −1 . . . −25), TRP5.i (5′−127 . . . −47)and TRP5.j (5′+128 . . . +48).

[0323] IROs used to generate the G270A mutation in SIR2 were 270.a(5′−70 . . . −1, +1 . . . +10), 270.b (5′+70 . . . +1, −1 . . . −10),270.e (5′−48 . . . −1, +1 . . . +47), 270.f (5′+47 . . . +1, −1 . . .−48); IROs used to generate the N345A mutation in SIR2 were 345.a (5′−85. . . −1, +1 . . . +10) and 345.b (5′+70 . . . +1, −1 . . . −10).

[0324] IROs used to generate the H364Y mutation in SIR2 were 364.a(5′−70 . . . −1, +1 . . . +10) and 364.b (5′+85 . . . +1, −1 . . . −10).

[0325] IROs used to generate the K40A mutation in RAD50 were RAD50.a(5′−70 . . . −1, +1 . . . +10) and RAD50.b (5′+70 . . . +1, −1 . . .−10).

[0326] IROs used to generate the D16A mutation in MREll were MRE.a(5′−69 . . . −1, +1 . . . +11) and MRE.b (5′+71 . . . +1, −1 . . . −9).

[0327] Cells from each transformation were spread on a single YPD plate,incubated at 30° C. and replica-plated to 5-FOA the following day. Afterthree days, colonies were replica-plated to YPD, G418, Trp⁻ (for TRP5experiments) and YPG (to select against petite mutants) plates.

Results

[0328] Deletion of Genomic Sequences

[0329] The delitto perfetto strategy was used to delete the entire MLP2gene from the start to the stop codon (FIG. 2A), such that noheterologous DNA was retained after the deletion process was complete.The CORE-cassette was targeted by standard homologous recombinationtechniques into three commonly used yeast strain backgrounds (BY4742,VL6α and E133) so as to replace the MLP2 ORF. This generated cells thatwere Ura+(5-FOA sensitive) and G418 resistant.

[0330] Two 80-nucleotide (nt) IROs (designated MLP2.a and MLP2.b) weredesigned with a 20 base overlap at their 3′ ends. Annealing and in vitroextension of MLP2.a and MLP2.b resulted in a 140-bp double-strandmolecule that contains 70 bp homologous to the sequence upstream anddownstream regions of the CORE-cassette. Cells were transformed withthese double-stranded molecules.

[0331] As shown in Table 1, the frequency of 5-FOA resistant(5-FOA^(R)), G418 sensitive (G418^(s)) cells was about 100transformants/0.5 nmoles of IRO DNA (35-45 μg) added per 10⁷ viablecells. All the G418^(s) clones tested by colony PCR had lost thecassette and were ΔMLP2. Since about half of the 5-FOA^(R) clones wereG418 resistant (presumably due to mutations in the KlURA3-encodingsequence), G418 sensitivity was diagnostic of CORE-cassette loss. Three5-FOA^(R), G418^(s) BY4742 isolates were sequenced and each onecontained the correct deletion (ΔMLP2). One of the isolates had acquireda point mutation in the 140-bp IRO region. TABLE 1 Accuracy of targetedchanges with delitto perfetto Correctly Clones w/o Designed IRO species5-FOA^(R)/ G418⁵/ Correctly targeted additional Strain Locus changeused^(a) 10⁷ cells^(b) 10⁷ cells^(c) excised CORE^(d) clones^(e)changes^(f) BY4742 MLP2 Δ ORF MLP2.a+b Pfx 225  97 18/18 3/3 2/3 VL6αMLP2 Δ ORF MLP2.a+b Pfx 215 118 6/6 ND ND E133 MLP2 Δ ORF MLP2.a+b Pfx284 101 6/6 ND ND E133 POL30 Δ ORF PCNA.a+b Pfx  70^(g)  4^(g)  9/12 NDND BY4742 TRP5 A334A TRP5.a+b Pfx 190 130 50/50 50/50 19/20 (BamHI)TRP5.e+f 286 212 40/40 40/40 11/15 BY4742 SIR2 G270A 270.a+b Pfx 302 10520/20 20/20 13/20 270.a+b 151  27 20/20 20/20 17/20 270.e+f 237 15019/19 19/19 16/19 BY4742 SIR2 N345A 345.a+b Pfx 263 126 11/11  9/11 6/11 BY4742 SIR2 H364Y 364.a+b Pfx 210  42 14/14 12/14  6/14 VL6α RAD50K40A RAD50.a+b Pfx 169  7 6/6 3/3 2/3 VL6α MRE11 D16A MRE11.a+b Pfx 205 6 6/6 3/3 3/3

[0332] Deletion of Essential Genes

[0333] The delitto perfetto approach was next applied to the deletion ofessential genes by integrating the CORE-cassette downstream from thegene. The CORE-cassette was integrated beyond the stop codon of thePOL30 gene (PCNA) (FIG. 2B) in strain E133, and a wild-type copy of thegene on a plasmid (pBL230) was introduced into the cell. Cells were thentransformed with the overlapping IROs PCNA.a and PCNA.b that hadhomology upstream of the start codon of the POL30 gene and downstream ofthe CORE-cassette.

[0334] Among twelve 5-FOA^(R) G418^(s) colonies obtained in threeexperiments, nine had a precise deletion of the chromosomal POL30 gene(Table 1). The reduced frequency of CORE-cassette replacement by IROs,as compared to the MLP2 deletion embodiment described above, likely isdue to the recombination competition with the plasmid-bome copy ofPOL30. Loss of the CORE-cassette was usually associated with areplacement event and not due to recombination with the plasmid.

[0335] The deletion clones generated in this manner were unable to losethe POL30 plasmid after over 30 generations in nonselective medium,whereas the plasmid was readily lost in control cells containing thewild-type chromosomal POL30 gene.

[0336] Site-Specific Mutagenesis

[0337] The delitto perfetto strategy was also applied to the rapidcreation of site-specific mutations in the genome (FIG. 1). Oneembodiment of this method was demonstrated by the introduction of asilent mutation in the TRP5 gene, which mutations generate a new BamHIsite (FIG. 2C).

[0338] The CORE-cassette was targeted into the TRP5 gene of strainBY4742 between nt 1002 and 1003, resulting in Trp⁻ cells(BY4742-TRP5-CORE). The resultant cells were transformed with the two80-nt IROs TRP5.a and TRP5.b containing the BamHI mutation site adjacentto the CORE-cassette insertion site, and subsequently 5-FOA^(R) G418^(s)colonies were isolated (Table 1). Among 50 clones tested, all had lostthe CORE-cassette and acquired the desired targeted mutation. Of 20G418^(s) clones sequenced, 19 did not have additional mutations.

[0339] Thus, embodiments of the delitto perfetto mutagenesis techniquecan be used for the efficient creation of site-specific changes using,for instance, IROs with a 20-bp overlap.

[0340] Multiple Rounds of Site-Specific Mutagenesis

[0341] Once the CORE-cassette is integrated into a sequence, multiplesite-specific modifications can be generated simply by designing newoligonucleotides and repeating the IRO transformation procedure. Todemonstrate this, the delitto perfetto strategy was applied tosite-specific changes in the regions surrounding two positions ofCORE-cassette integration (codon 270 and between codons 354 and 355) inthe SIR2 gene (FIG. 2D and 2E). Mutations were contained in either theoverlapping regions (IROs 270.a+b) or in just one of the pairoligonucleotides (345.a+b, and 364.a+b).

[0342] Following annealing and DNA synthesis (with Pfx polymerase), thestrains were transformed using IRO pairs 270.a+b and 345.a+b, or364.a+b, respectively. As expected, all 5-FOA^(R) G418^(s) clones hadlost the CORE-cassette, based on colony PCR (Table 1). The site-directedmutation targeting was highly efficient. For the IROs containing amutation near the CORE-cassette, all clones had acquired the correctchange (20/20), as shown in Table 1. For the IROs with a mutationexternal to the original oligonucleotide overlap, over 80% of the cloneshad the correct site-specific change (21/25).

[0343] Site-Directed Mutagenesis Using a Single IRO

[0344] Site-directed mutagenesis can be accomplished by addingindividual oligonucleotides directly to cells, rather than pairs ofoligonucleotides. Single IROs were compared for their ability to createsite-specific mutations when added directly to cells. The strainBY4742-TRP5-CORE was transformed with several IROs (FIG. 3) that wouldyield the silent BamHI mutation in TRP5 (FIG. 2C). The 95 nt (e or f)and 81 nt (e1 or f1) single IROs that extend to either side of theCORE-cassette were comparably effective at generating site-directedmutations when added individually (FIG. 4A). The minimum length ofhomology for efficient mutagenesis was between 25 and 40 nt (compare the51-mers e2 and f2 with e1 and f1).

[0345] Site-Directed Mutagenesis Using Various Pairs of IROs

[0346] Combinations of IROs were also examined for their ability tocreate the same site-specific mutation. Individual IROs were addeddirectly to competent cells without in vitro annealing or extension. Asshown in FIG. 4B, modification by unannealed pairs of 81 or 95-nt IROswas highly efficient if they were fully complementary; the frequencieswere even higher than those for oligonucleotides annealed and extendedin vitro. As observed with single IROs, the minimum homology requiredfor efficient mutagenesis was between 25 and 40 nt (e2+f2 vs. e1+f1).

[0347] Surprisingly, the combination of TRP5.a+b, having only a 20 bp3′-overlap, was also able to create the site-specific change at a levelabout one-sixth of that found for these pairs of DNA oligonucleotideswhen annealed and extended in vitro (TRP5.a+b Pfx). A similar pair ofoligonucleotides, but with opposite polarity resulting in 5′-overlaps(c+d in FIG. 3) yielded no transformants.

[0348] TRP5.e+j or TRP5.f+i, which do not overlap, showed notransformation increase over either TRP5.e or TRP5.f alone. Similarresults were obtained for modifications at SIR2 codon 270 (see FIG. 2D)in strain BY4742 using corresponding single and double IROs (270.a, b,c, d, e, f, e1, f1, e2, f2, j and i).

[0349] Site-specific targeting by complementary IROs was highlyefficient; after positive/negative selection nearly all the isolates hadlost the CORE-cassette and acquired the desired mutation. Confirmationof successful mutation is greatly simplified because only the regioncovered by the oligonucleotides needs to be sequenced in a small numberof samples; mutations outside this region were never detected. Forexample, among the clones that were sequenced for the TRP5 (A334A-BamHI)and SIR2 G270A changes, 5-35% associated point mutations were found inthe IRO region when complementary oligonucleotides were extended invitro or were used directly (Table 1).

[0350] While a single IRO could be used for mutagenesis, the efficiencywas much greater with pairs of overlapping IROs. In addition, thetargeting efficiency was greater as the size of single and complementaryIROs was increased (FIG. 4A and 4B). This differs from a previous reportusing single-stranded oligonucleotides (Moerschell et al., Proc. Natl.Acad. Sci. USA 85, 524-528, 1988), which system did not utilize acounterselection scheme that requires elimination of a large region(i.e., the CORE-cassette). Possibly, the reliance of delitto perfettomutagenesis on a recombinational pathway (see below) accounts for thedifference.

[0351] In certain embodiments, the efficiency of the delitto perfettomethod was greatest when fully complementary oligonucleotides thatwere≧81 nt long were used (FIG. 3 and FIG. 4B). The two pairs of IROsthat gave the highest integration efficiencies were complementary95-mers e+f and extended a+b oligomers (a+b Pfx) that could yield a 140double-strand molecule. IROs such as e+f appear to be the most practicalfor introducing specific point mutations anywhere within a 15-nt regionand can be used to create small or large deletions as described in FIGS.1 and 5A. Pairs of IROs with no overlap were no more effective thanusing the respective individual IROs. DNAs with 3′-end overlaps weremoderately efficient even without in vitro annealing and extension;however, IROs that had overlapping 5′-ends were not productive. Thissuggests that strands with as few as 20 overlapping nucleotides cananneal during the transformation procedure and lead to efficientsite-specific mutation through a process that probably involves DNAsynthesis. However, it is possible that the annealed structure interactsdirectly with chromosomal DNA. It also appears that there is little DNAdegradation within the cell since protected oligonucleotides that werephosphorothioated at their 3′ and 5′ ends did not increase thetransformation efficiency of the IRO pairs (TRP5.a+b, TRP5.c+d, orTRP5.e+f).

[0352] As demonstrated with the SIR2-N345A and H364Y mutations, once theCORE-cassette is inserted at a particular location, the DNA can bemodified with different pairs of IROs at distances that span the CORE atleast 30 bp upstream and downstream without moving the cassette. Basedon the reported results, it is believed that specific mutations can becreated up to 100 nt from a CORE-cassette using 100-nt IROs with a 20-ntoverlap, as shown in FIG. 5C. This is a significant advance over otherapproaches for the study of specific regions since in all othermutagenesis procedures each step in the creation of site-specificmutations must be repeated from the beginning, even for additionalmodifications of the same residue.

[0353] Additional mutations found within targeted clones were neverdetected outside the sequence covered by IROs. The spectrum ofadditional mutations within the sequence replaced by IROs included 70%deletions and was as follows: 46.7% 1-bp deletions, 26.7% 1-bpsubstitutions, 16.7% 2-bp deletions, 6.6% 1-bp deletions plus single bpsubstitutions and 3.3% duplications of part of an IRO sequence.

Example 2 Targeting by IROs in Delitto Perfetto Involves HomologousRecombination

[0354] Since the RAD52 gene is important for nearly all types ofhomologous recombination, its role in delitto perfetto mutagenesis wasexamined. Wild-type and mutant (RAD52::LEU2; two independent isolates)strains were transformed with a control centromeric plasmid pLKL67Y(containing the HIS3 and URA3 genes), or the following TRP5 IROs: (I)a+b Pfx, (2) a+b, (3) c+d, (4) e+f, (5) e and (6) f.

[0355] In repeated transformations with pLKL67Y, the wild-typetransformation level was about 4-fold higher than for the rad52 mutant(4.3×10⁵/μg vs. ˜1×10⁵/μg, respectively), apparently due to reducedplating efficiency in the rad52 mutant. No 5-FOA^(R) G418^(s) cloneswere observed in the rad52 strain with various IROs in three separateexperiments even when 10 times more e+f was used (FIG. 4A and 4B).Complete RAD52 dependence was also observed for modifications at SIR2codon 270 (see FIG. 2D) using IROs 270.a+b Pfx, 270.a+b, and 270.c+d.These results indicate that oligonucleotide replacement during delittoperfetto mutagenesis is mediated by a recombinational mechanism.

[0356] The importance of the RAD50 and MREll genes, which function inrecombination as well as double-strand break end-joining, on IROsite-directed changes was examined also. The CORE-cassette wasintegrated in strain VL6α into each gene, at positions described inFIGS. 2F and G. IROs were created that produce an AA to CG substitutionin RAD50 at codon 40 and an A-to-C transversion in codon 16 of MREll.Even though the efficiency of removal of the CORE-cassette was low withthe respective a+b Pfx IROs, several G418^(s) colonies were obtainedthat had a correct excision of the CORE-cassette (Table 1). Sequenceanalysis of three G418^(s) isolates for both RAD50 and MREll showed thatthe expected modifications had occurred in all clones and only oneisolate (of RAD50) displayed an additional point mutation. Thus, theoligonucleotide integration process is RAD52-dependent at the level ofmutation detection used (contrast with delitto perfetto-DSB, below), butthere appears to be less dependence on the RAD50 and MREll genes.

[0357] The integration of IROs, whether presented singly or as pairs tocells, was completely dependent on the RAD52 gene in this example, andappeared to be partially dependent on RAD50 and MREll. The relativelymoderate effect of the latter genes is consistent with their having asmaller effect on recombination (Lewis and Resnick, Mutat. Res.451:71-89, 2000). While they have been implicated in DNA end-joining,the absence of an effect on IRO-mediated integration in sir2 mutants,which are deficient in end-joining (Lewis and Resnick, Mutat. Res.451:71-89, 2000), suggests that their role in delitto perfetto isspecific to recombination.

[0358] The minimum amount of homology required for efficientrecombination at the ends of transforming molecules in yeast is about 30bp (Manivasakam et al., Nucleic Acids Res. 23:2799-2800, 1995).Similarly, results using delitto perfetto mutagenesis suggest that theminimum length of homology in IROs must be between 25 and 40 nt.

[0359] In contrast to the methods reported herein, the DNA-RNA chimericoligonucleotide mutagenesis system in higher eukaryotes is not mediatedby homologous recombination (Cole-Strauss et al., Nucleic Acids Res.27:1323-1330, 1999; Yoon et al., Proc. Natl. Acad. Sci. USA93:2071-2076, 1996), nor does the oligonucleotide approach in yeastdescribed by Moerschell et al., Proc. Natl. Acad. Sci. USA 85, 524-528,1988 (see also Erdeniz et al., Genome Res. 7, 1174-1183, 1997; Yamamotoet al., Yeast 8:935-948, 1992). The delitto perfetto system differs fromthose previously described at least in that small nucleic acid regionsdistant from each other (separated by the CORE-cassette) are broughtinto close proximity to be paired with the oligonucleotide sequences.This is believed to account for the requirement for the RAD52 epistasisgroup proteins.

Example 3 A Diagnostic Tool for Assessing Tumor Associated p53 Mutations

[0360] An expression cassette containing the human p53 coding sequenceunder the GAL1 promoter is integrated into a suitable chromosomal locus(e.g., LYS2 gene on chromosome III ) of a haploid yeast strain. Asdescribed in FIG. 1, the CORE-cassettes were targeted by transformationinto seven different positions in the p53 coding sequence at 95nucleotide intervals across the DNA binding region, from nucleotide 315to nucleotide 885. Thus, a panel of seven isogenic yeast strainscontaining one CORE-cassette at each of the indicated positions werecreated. Oligonucleotides containing desired p53 mutations were designedto replace by transformation the closest CORE-cassette in theappropriate strain resulting in the appropriate site-directed mutation.Changes are confirmed by in vitro amplification (e.g., PCR) and sequenceanalysis of the p53 coding sequence region containing the introducedmutation.

[0361] The consequences of the human associated p53 mutations can beexamined by expressing each mutated p53 molecule and examining theability to induce transcription at various p53 responsive elements,corresponding to p53 responsive genes. Systems for addressing in vivop53 affinity/specificity are described in, for instance, Inga et al.(Oncogene 20:501-513, 2001); and in Inga and Resnick (Oncogene20:3409-3419, 2001).

[0362] This is a demonstration of integrating the CORE-cassette atpositions corresponding to the site between amino acid 26 and 27 in p53.A set of oligonucleotides were used to create a variety of mutations ator near this site. Using that set of CORE and oligonucleotides,mutations were made in Ser 15, 20, 33, 37, 47 and Thr 18 creatingalanine or aspartate changes.

Example 4 Alternative CORE-cassettes

[0363] Four alternative CORE-cassettes were constructed in the basicpFA6a E. coli vector, in order to expand the applicability of thedelitto perfetto approach to every yeast strain (also to URA⁺ and G₄₁₈^(R) strains), including wild type strains. Two different heterologousmarkers were considered, a reporter that provides resistance tohygromycin and a new countereslectable marker, which is a variant of thep53 protein (V122A). When the variant p53 is highly expressed under aninducible GAL1/10 promoter, growth of yeast is prevented. By acombination of these markers with the previous markers (KlURA3 andkanMX4) four new cassettes were made: CORE-UK (KlURA3 and kanMX4),CORE-UH (KlURA3 and HygroR), CORE-KpS3 (kanMX4and GAL-p53) and CORE-Hp53(HygroR and GAL-p53). Similar CORE-cassettes can be used with otherselectable and counterselectable genes.

Experimental Protocols

[0364] Yeast Strains and Growth Conditions

[0365] Yeast strains used were: BY4742, and VL6α. Cells were grown instandard rich (YPD), glycerol (YPG) and synthetic minimal medium withouturacil (SD Ura⁻), or tryptophan (SD Trp⁻). Geneticin (G418) resistantcells were grown on YPD plates containing 200 μg/ml of G418. Hygromycinresistant cells were grown on YPD plates containing 200 μg/ml ofhygromycin (Invitrogen, Carlsbad, Calif.). Ura cells were selected onsynthetic complete medium containing 5-fluoroorotic acid (5-FOA). Cellsthat have lost the CORE cassette containing the p53 gene were selectedon synthetic complete media containing 2% galactose in place of glucose(GAL) (Sherman et al. (eds.) Methods in Yeast Genetics, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0366] Plasmids

[0367] The plasmids containing the new CORE-cassettes were termed:pCORE-UK, pCORE-UH, pCORE-Kp53, pCORE-Hp53. Plasmid pCORE-UK wasconstructed by cloning a 1.5 kb SacI-SmaI fragment containing the kanMX4gene (Wach et al., Yeast 10, 1793-1808, 1994) into the SacI-HpaI sitesof pFA6aKlURA3 (Delneri et al., Yeast 15:1681-1689, 1999). The plasmidpCORE-UH was constructed by cloning a 1.7 kb SacI-SmaI fragmentcontaining the hygromicin resistance (HygroR) gene from the pAG32 vector(Goldstein and McCusker, Yeast 14, 1541-1553, 1999) into the SacI-HpaIsites of pFA6aKlURA3. pCORE-Kp53 was constructed by cloning a 2.1 kbEcoRI-SpeI fragment containing the p53 V 122 gene under the GAL1/10promoter (Inga and Resnick, Oncogene 20, 3409-3419, 2001) into theEcoRI-SpeI sites of pFA6akanMX4 (Wach et al., Yeast 10, 1793-1808,1994). pCORE-Hp53 was constructed by cloning a 1.7 kb SacI-BamHIfragment containing the HygroR gene from pAG32, into the SacI-BgIIIsites of pCORE-Kp53, replacing the kanMX4 gene.

[0368] PCR amplification and sequence analysis

[0369] The CORE-UK, -UH, -Kp3 and -Hp53 cassettes were amplified as a3.2, 3.5, 3.7 and 4.0 kb DNA fragment, respectively from thecorresponding vectors using Taq DNA polymerase (Roche, Indianapolis,Ind.), with 32 cycles of 30 seconds at 94° C., 30 seconds at 56° C., and4 minutes at 72° C.

[0370] For integration of the CORE-cassettes into chromosomal loci,chimeric 70-mers were designed, consisting of 50 nucleotides homologousto the appropriate flanking region of the genomic target locus and 20nucleotides, shown here, which allow for the amplification of all thefour CORE-cassettes (these oligos do not work to amplify the originalCORE cassette):

[0371] 5′- . . . TTCGTACGCTGCAGGTCGAC-3′ for the KlURA3 side in pCORE-UKand pCORE-UH and for the kanMX4 or HygroR side in pCORE-Kp53 andpCORE-Hp53, respectively (entire oligonucleotide shown in SEQ ID NO: 61)and

[0372] 5′- . . . CCGCGCGTTGGCCGATTCAT-3′ for the kanMX4 or HygroR sidein pCORE-UK and pCORE-UH, respectively, and for the GAL1/10-p53 side inpCORE-Kp53 and pCORE-Hp53 (entire oligonucleotide shown in SEQ ID NO:62).

[0373] Amplification using these 70-mers yielded PCR product with 50nucleotides each of the upstream and downstream chromosomal sequence,flanking the CORE-cassettes themselves. Initial insertion of theCORE-cassettes into gene the TRP5gene was performed using the followingprimers: TRP5.I/TRP5.II (SEQ ID NOs: 61 and 2). K1 (SEQ ID NO: 74), H1(SEQ ID NO: 76), URA3.1 (SEQ ID NO: 75) and p7 (SEQ ID NO: 77) areinternal primers in the CORE-cassettes, within kanMX4, HygroR, KlURA3and GAL1/10-p53, respectively.

[0374] CORE-UK insertion was verified at the TRP5 locus using thefollowing primer combinations: TRP5.1/URA3.1 (SEQ ID NOs: 21 and 75) andTRP5.2/K1 (SEQ ID NOs: 22 and 74). CORE-UH insertion was verified at theTRP5 locus using TRP5.1/URA3.1 and TRP5.2/H 1. CORE-Kp53 insertion,using TRP5.1/K.1 (SEQ ID NOs: 21 and 74) and TRP5.2/p7 (SEQ ID NOs: 22and 74); and CORE-Hp53 insertion, using TRP5.1/H.1 (SEQ ID NOs: 21 and76) and TRP5.2/p7 (SEQ ID NOs: 22 and 77). CORE-cassettes excision andsequence analysis was carried out using the following primers:TRP5.1/TRP5.2 (SEQ ID NOs: 21 and 22).

[0375] Integrative Recombinant Oligonucleotide (IRO) Mutagenesis

[0376] IROs used to generate the silent mutation in the codon 334 ofTRP5 were TRP5.e (5′−47 . . . −1, +1 . . . +48; SEQ ID NO: 35),TRP5.f(5′+48 . . . +1, −1 . . . 47; SEQ ID NO: 36).

[0377] Cells from each transformation were spread on a single YPD plate,incubated at 30° C. and replica-plated to 5-FOA or GAL plates thefollowing day. Cells growing on GAL plates were replica-plated on freshGAL plates every day, to reduce the background growth. After three days,colonies were replica-plated from 5-FOA or GAL to YPD, G418, Trp⁻ andYPG (to select against petite mutants) plates.

[0378] Results: Alternative CORE-cassettes and the use of p53 as aCounterselectable Marker

[0379] Four additional CORE-cassettes were constructed: CORE-UK (KlURA3and kanMX4), CORE-UH (KlURA3 and HygroR), CORE-Kp53 (kanMX4and GAL-p53)and CORE-Hp53 (HygroR and GAL-p53). All cassettes were tested in thedelitto perfetto strategy for the rapid creation of site-specific andsilent mutations in the TRP5 gene, where the mutations generate a newBamHI site, as described above. The CORE-cassette s were targeted intothe TRP5 gene of strain BY4742 and VL6α between nt 1002 and 1003,resulting in Trp⁻ cells (BY4742-TRP5-CORE, and VL6α-TRP5-CORE). Theresultant cells were transformed with the two 95-nt TRP5.e and TRP5.fcontaining the BamHI mutation site, and subsequently 5-FOA resistant,G418^(s) or HygroS colonies were isolated (Table 2). TABLE 2 Efficacy ofthe different CORE-cassettes in delitto perfetto mutagenesis 5- IROFOA^(R)/ G418⁵/ Designed species 10⁷ 10⁷ Strain^(a) Locus changeused^(b) cells^(c) cells^(d) BY4742-UK TRP5 A334A (BamHI) TRP5.e+f 315177 BY4742-UH TRP5 A334A (BamHI) TRP5.e+f 259 120 BY4742- TRP5 A334A(BamHI) TRP5.e+f 366 52 Kp53 BY4742- TRP5 A334A (BamHI) TRP5.e+f 369 53Hp53 VL6α-UK TRP5 A334A (BamHI) TRP5.e+f 115 33 VL6α-UH TRP5 A334A(BamHI) TRP5.e+f 88 30 VL6α-Kp53 TRP5 A334A (BamHI) TRP5.e+f 59 8VL6α-Hp53 TRP5 A334A (BamHI) TRP5.e+f 93 23

[0380] Four independent G418_(s) or HygroS clones from each strain thatwere tested for the presence of the BamHI site demonstrated that theoligo targeting was correct. Thus, the p53 allele V122A can be used as ageneral counter-selectable marker.

[0381] Though illustrated here using the CORE-cassette format, thecassettes produced in this example could equally be used in CORE-DSB-and RE-DSB-cassette formats, simply by changing other elements in thecassette. This alternative embodiments are specifically contemplated andthereby included in this disclosure.

Example 5 Delitto Perfetto-DSB Mutagenesis

[0382] This example provides a representative description of delittoperfetto-DSB mutagenesis, in which an expression inducible double-strandbreak enzyme and corresponding double-strand break site are introducedinto a cassette that contains a reporter marker (RE-DSB) or acounterselectable and a reporter marker (CORE-DSB). Induction andresultant expression of the double-strand break enzyme prior tointroduction of the IRO stimulates double-strand breaks at thecorresponding site, which results in very high frequencies of IROmediated changes.

Experimental Protocols

[0383] Yeast strains and growth conditions

[0384] Yeast strains used were: BY4742 and E133a (S1-A12: MATα, ade5-1,lys2-12A, trp1-289, his7-2, leu2-3,112, ura3-52; unpublished), Cellswere grown in standard rich (YPD), glycerol (YPG) and synthetic minimalmedium without uracil (SD Ura⁻), or tryptophan (SD Trp⁻). Geneticin(G418) resistant cells were grown on YPD plates containing 200 μg/ml ofG418. Hygromycin resistant cells were grown on YPD plates containing 200μg/ml of hygromycin. Ura⁻ cells were selected on synthetic completemedium containing 5-fluoroorotic acid (5-FOA). . Cells beforetransformation with oligonucleotides were grown in liquid syntheticcomplete media containing 0.02, 0.2 or 2% galactose in place of glucose(GAL).

[0385] Plasmids

[0386] The plasmids containing the CORE -DSB cassette were termed:pCORE-GALSceKU and pCORE-GALSceHU. Plasmid pCORE-GALSceKU andpCORE-GALSceHU were constructed by cloning a 1.4 kb BglII fragmentcontaining the I-SceI gene under the GAL1/10 promoter into the BglIIsite of pCORE-UK and pCORE-UH. The 1.4 kb fragment containing the I-SceIgene under the GAL1/10 promoter was obtained by PCR amplification fromthe pWY203 vector (Galli and Schiestl, Genetics 149, 1235-1250, 1998)using the following primers: Sce.FI and Sce.R containing the BglII site.

[0387] PCR amplification and sequence analysis. The RE-DSB cassettesderived from pCORE-GALSceKU and pCORE-GALSceHU were amplified as a 2.7and 3.0 kb DNA fragment, respectively from the corresponding vectorsusing Taq DNA polymerase (Roche, Indianapolis, Ind.), with 32 cycles of30 seconds at 94° C., 30 seconds at 56° C., and 3 minutes at 72° C.

[0388] For integration of the short CORE-cassettes into chromosomalloci, one chimeric 70-mer and one chimeric 88-mer containing also theI-SceI site were designed, consisting of 50 nucleotides homologous tothe appropriate flanking region of the genomic target locus and 20 and38 nucleotides, shown here, which allow for the amplification of all thefour CORE-DSB cassettes:

[0389] 5′- . . . CATCTGGGCAGATGATGTCG-3′ for the kanMX4 or the HygroRside (entire oligonucleotide shown in SEQ ID NO: 70) and

[0390] 5′- . . . TAGGGATAACAGGGTAATTTGGATGGACGCAAAGAAGT-3′ for theGAL1/10::I-SceI side (the I-SceI site is in bold) (entireoligonucleotide shown in SEQ ID NO: 65).

[0391] The CORE-DSB cassettes from pCORE-GALSceKU and pCORE-GALSceHUwere amplified as a 4.6 and 4.9 kb DNA fragment, respectively from thecorresponding vectors using Taq DNA polymerase (Roche, Indianapolis,Ind.), with 32 cycles of 30 seconds at 94° C., 30 seconds at 56° C., and4 minutes and 30 seconds at 72° C.

[0392] For integration of the CORE-cassettes into chromosomal loci, onechimeric 70-mer and one chimeric 88-mer containing also the I-SceI sitewere designed, consisting of 50 nucleotides homologous to theappropriate flanking region of the genomic target locus and 20 and 38nucleotides, shown here, which allow for the amplification of all thefour CORE-DSB cassettes:

[0393] 5′- . . . TTCGTACGCTGCAGGTCGAC-3′ for the KlURA3 side (entireoligonucleotide shown in SEQ ID NO: 61) and

[0394] 5′- . . . TAGGGATAACAGGGTAATTTGGATGGACGCAAAGAAGT-3′ for theGAL1/10::I-SceI side (the I-SceI site is in bold) (entireoligonucleotide shown in SEQ ID NO: 65).

[0395] Initial insertion of the RE-DSB (I-SceI site+GAL1−I-SceI+kanMX4)or (I-SceI site+GAL1−I-SceI+HygroR) cassette into the TRP5 gene wasperformed using the following primers: TRP5.IK/TRP5.SceII. The I-SceIsite can be placed on both sides of the cassette and in differentorientations: TSce.IK/TRP5.II or TecS.IK/TRP5.II, or two inverted I-SceIsites can be placed together at one side of the cassette:TRP5.IK/TscecS.II, or two I-SceI sites can be placed at both sides ofthe cassette in direct (TSce.IK/TRP5.SceII) or opposite orientation(TecS.IK/TRP5.SceII) for both cassettes.

[0396] Amplification using these primers yielded PCR product with 50nucleotides each of the upstream and downstream chromosomal sequence,flanking the CORE-DSB cassettes themselves. K.2, H.2 and Sce.2 areinternal primers in the RE-DSB cassettes, within kanMX4, HygroR andGAL1/10I-SceI, respectively.

[0397] RE-DSB cassette insertion was verified at the TRP5 locus usingthe following primer combinations: TRP5.1/K2 or TRP5.1/H2 andTRP5.2/Sce.2.

[0398] CORE-cassette excision was carried out using the followingprimers: TRP5.1/TRP5.2.

[0399] Integrative Recombinant Oligonucleotide (IRO) Mutagenesis

[0400] IROs used: TRP5.e (5′−47 . . . −1, +1 . . . +48), TRP5.f(5′+48 .. . +1, −1 . . . −47), TRP5.e1 (5′40 . . . −1, +1 . . . +41), TRP5.f1(5′+41 . . . +1, −1 . . . −40), TRP5.e2 (5′−25 . . . −1, +1 . . . +26),TRP5.f2 (5′+26 . . . +1, −1 . . . −25), TRP5.e3 (5′−20 . . . −1, +1 . .. +21), TRP5.f3 (5′+21 . . . +1, −1 . . . −20), TRP5.e4 (5′−15 . . . −1,+1 . . . +16), TRP5.f4 (5′+16 . . . +1, −1 . . . −15).

[0401] Cells before transformation were grown for 20 minutes to 5 hoursin liquid GAL in order to induce I-SceI endonuclease expression. Theenzyme targets its site in the cassette and generates a DBS. Cells fromeach transformation were spread directly on Trp⁻ plates, or on YPDplates, incubated at 30° C.

[0402] Initial insertion of the CORE-DSB (I-SceIsite+GAL1−I-SceI+kanMX4+KlURA3) or (I-SceIsite+GAL1−I-SceI+HygroR+KlURA3) cassette into the TRP5 gene wasperformed using the following primers: TRP5.1/TRP5.SceII. The I-SceIsite can be placed on both sides of the CORE and in differentorientations: TSce.IU/TRP5.II or TecS.IU/TRP5.II, or two inverted I-SceIsites can be placed together at one side of the CORE: TRP5.I/TscecS.II,or two I-SceI sites can be placed at both sides of the CORE in direct(TSce.IU/TRP5.SceII) or opposite orientation (TecS.IU/TRP5.SceII) forboth cassettes.

[0403] Amplification using these primers yielded PCR product with 50nucleotides each of the upstream and downstream chromosomal sequence,flanking the CORE-DSB cassettes themselves. URA3.1 and Sce.2 areinternal primers in the CORE-DSB cassettes, within KlURA3 andGAL1/10I-SceI.

[0404] CORE-DSB cassettes insertion was verified at the TRP5 locus usingthe following primer combinations: TRP5.1/URA3.1 and TRP5.2/Sce.2.

[0405] CORE-cassettes excision was carried out using the followingprimers: TRP5.1 /TRP5.2.

[0406] Integrative Recombinant Oligonucleotide (IRO) Mutagenesis

[0407] IROs used: TRP5.e (5′−47 . . . −1, +1 . . . +48), TRP5.f(5′+48 .. . +1, −1 . . . 47), TRP5.e1 (5′−40 . . . −1, +1 . . . +41), TRP5.f1(5′+41 . . . +1, −1 . . . −40), TRP5.e2 (5′−25 . . . −1, +1 . . . +26),TRP5.f2 (5′+26 . . . +1, −1 . . . −25). Additional IROs used to generatethe silent mutation in the codon 334 of TRP5 were TRP5.e3 (5′−20 . . .−1, +1 . . . +21), TRP5.f3 (5′+21 . . . +1, −1 . . . −20),TRP5.e4(5′−15. . . −1, +1 . . . +16), TRP5.f4 (5′+16 . . . +1, −1 . . . −15).

[0408] Cells before transformation were grown for 20 minutes to 5 hoursin liquid GAL, in order to induce I-SceI endonuclease expression. Theenzyme targets its site in the cassette and generates a DBS. Cells fromeach transformation were spread directly on Trp⁻ plates, or on YPDplates, incubated at 30° C. and replica-plated to 5-FOA plates thefollowing day. After three days, colonies were replica-plated from 5-FOAor GAL to YPD, G418, Trp⁻ and YPG (to select against petite mutants)plates.

[0409] Results: Delitto Perfetto-DSB System

[0410] The delitto perfetto-DSB strategy was applied to the highthroughput creation of site-specific mutations in the genome in the TRP5locus, by the introduction of a silent mutation in the TRP5 gene, wherethe silent mutation generates a new BamHI site.

[0411] The RE-DSB and CORE-DSB cassettes were targeted into the TRP5gene of strain BY4742 between nt 1002 and 1003, resulting in Trp⁻ cells(BY4742-TRP5-CORE). Cells containing the RE-DSB were transformed withoutand with the induction of DSB (5 hours in GAL 2%) with tvo 96-nt IROsTRP5.e and TRP5.f, or 51-mres TRP5.e2 and TRP5.f2, or 41-mers TRP5.e3and TRP 5f3, or 31-mers TRP5.e4 and TRP5.f4 containing the BamHImutation site adjacent to the cassette insertion site, and subsequentlyTrp⁺ colonies were isolated (Table 3). TABLE 3 Frequency of IROmutagenesis after DSB induction viable DSB Trp⁺ Trp⁺ clones InductionIRO species Trp⁺ clones clones Dil status^(a) used^(b) clones^(c) Dil10² x^(d) Dil 10⁴ x^(e) 10⁵ x^(f) No induction —   0 No inductionTRP5.e+f  417.25^(g)  3.75 282.25 No induction TRP5.e2+f2   40  52 Noinduction TRP5.e3+f3   0  53 No induction TRP5.e4+f4   0  33 induction —  0 induction TRP5.e+f  ˜10⁶ ˜10⁴ 185.75 480 induction TRP5.e2+f2 ˜5000429.3  3.67 406 induction TRP5.e3+f3   25.67 332 induction TRP5.e4+f4  0.3 322

[0412] Direct replica plating of the colonies grown on YPD (last colonon right of Table 3) on Trp⁻ media gave 2 to 5% Trp⁺ clones. This is theefficiency of targeting with IROs after induction of DSB.

Example 6 Deletion of Genomic Sequences

[0413] The delitto perfetto strategy (e.g., Example 1) was used todelete the up to 16 kb of genomic DNA, such that no heterologous DNA wasretained after the deletion process was complete. The originalCORE-cassette was targeted by standard homologous recombinationtechniques into BY4742 strain in the TRP5 locus as described above. Thisgenerated cells that were Ura+(5-FOA sensitive) and G418 resistant.

[0414] A series of four 60-mers (designated 1Stu.a, 1Stu.b, 2Stu.a and2Stu.b (SEQ ID NOs: 55-58, respectively)) were designed with a 20 baseoverlap at their 3′ ends containing also a StuI restriction site.Annealing and in vitro extension of 1Stu.a and 1Stu.b, 1Stu.a and2Stu.b, 2Stu.a and 1Stu.b, 2Stu.a and 2Stu.b resulted in a 100-bpdouble-strand molecule that contains 50 bp homologous to the sequenceupstream and downstream regions of the CORE-cassette. Cells weretransformed with these double-stranded molecules and transformants 5-FOAresistant G418 sensitive clones were isolated as described previously.1Stu.a and 1Stu.b are designed to make a16 kb deletion, IStu.a and2Stu.b are designed to make a 10.3 deletion, 2Stu.a and 1Stu.b aredesigned to make a 8 kb deletion, 2Stu.a and 2Stu.b are designed to makea 3.2 kb deletion aruond the TRP5 locus on chromosome VII.

[0415] The number (from three independent transformation events) of5-FOA resistant (5-FOA^(R)), G418 sensitive (G418^(s)) clones relativeto the different size deletions made using IROs are the following: 80.5for the 3.2 kb deletion, 27 for the 8 kb deletion, 3.5 for the 10.3 kbdeletion, and 2,5 for the 16 kb deletion. All the G418^(s) clones testedby colony PCR using CGR 1.1 and STT3.1 primers had lost the cassette andshowed the correct StuI restriction pattern.

[0416] Large deletions similar to those disclosed in this example alsocan be generated using the delitto perfetto-DSB system.

Example 7 Targeting by IROs using the Delitto Perfetto-DSB Approachwhere Homologous Recombination is Severely Impaired (e.g., rad52-nullBackground)

[0417] As discussed herein, in the delitto perfetto system, theintegration of IROs (whether presented singly or as pairs to cells)replacing the original CORE-cassette, was dependent on the RAD52 geneand appeared to be partially dependent on RAD50 and MREll. It has nowbeen surprisingly found that, once a CORE-DSB (or RE-DSB) cassette isplaced into the genome, the DSB-enhanced approach can be used even inthe absence of a highly efficient recombination systems.

[0418] Since the induction of a DSB in the RE-DSB cassette results in anextremely efficient targeting using IROs, the change in targetingefficiency was examined in the absence of the RAD52 gene. Wild-type andmutant (RAD52::LEU2; two independent isolates) strains were transformedwith a control centromeric plasmid Ycp50 (Rose et al., Gene, 60:237-243,1987) (containing the URA3 gene), or the following TRP5 IROs: TRP5.e+f,TRP5.e2+f2, TRP5.e3+f3 and TRP5.e4+f4.

[0419] In repeated transformations with YCp50, the wild-typetransformation level was about 2-3-fold higher than for the rad52mutant, apparently due to reduced plating efficiency in the rad52mutant. Complete RAD52 dependence was observed in the absence of DSBinduction. However, after DSB induction a high level of IRO targetingwas obeserved (Table 4). These results indicate that oligonucleotidereplacement during delitto perfetto-DSB mutagenesis can still occur inthe absence of RAD52, highlighting a residual level of homologousrecombination targeting independent from RAD52 function. TABLE 4Frequency of IRO targeting after DSB induction in a rad52-nullbackground DSB Induction IRO species Trp⁺ status^(a) Strain^(b) used^(c)clones^(d) No induction Δrad52 (1)/(2) — 0 No induction Δrad52 (1)/(2) TRP5.e+f 0 No induction Δrad52 (1)/(2) TRP5.e2+f2 0 No induction Δrad52(1)/(2) TRP5.e3+f3 0 No induction Δrad52 (1)/(2) TRP5.e4+f4 0 InductionΔrad52 (1) — 0 Induction Δrad52 (1)  TRP5.e+f 158.67 Induction Δrad52(1) TRP5.e2+f2 62.67 Induction Δrad52 (1) TRP5.e3+f3 2.3 InductionΔrad52 (1) TRP5.e4+f4 0.67 Induction Δrad52 (2) — 0 Induction Δrad52 (2) TRP5.e+f 242 Induction Δrad52 (2) TRP5.e2+f2 45.3 Induction Δrad52 (2)TRP5.e3+f3 0 Induction Δrad52 (2) TRP5.e4+f4 0

Example 9 Kits

[0420] The delitto perfetto and delitto perfetto-DSB mutagenesismethods, and components necessary or useful for carrying out suchmethods, disclosed herein can be supplied in the form of kits for use inperforming in vivo mutagenesis reactions. In such a kit, an amount ofone or more of the CORE-cassettes, CORE-DSB cassettes, RE-cassettes,and/or one or more IROs is provided in one or more containers. Thenucleic acid molecules may be provided suspended in an aqueous solutionor as a freeze-dried or lyophilized powder, for instance. Optionally thekits also include one or more appropriate yeast strains or cells ofother organisms.

[0421] Kits according to this invention can also include instructions,usually written instructions, to assist the user in performing in vivomutagenesis reactions with a CORE-cassette and/or one or more IROs. Suchinstructions can optionally be provided on a computer readable medium.

[0422] The container(s) in which the nucleic acid(s) are supplied can beany conventional container that is capable of holding the supplied form,for instance, microfuge tubes, ampoules, or bottles. In someapplications, the nucleic acid(s) may be provided in pre-measured singleuse amounts in individual, typically disposable, tubes or equivalentcontainers.

[0423] The amount of each CORE-cassette or oligonucleotide IRO (or pairof IROs) supplied in the kit can be any appropriate amount, dependingfor instance on the market to which the product is directed. Forinstance, if the kit is adapted for research or clinical use, the amountof each IRO provided likely would be an amount sufficient to mediateseveral transformation reactions. The examples illustrated hereinprovide guidelines for the amount of IROs useful for a singletransformation.

[0424] Similarly, in those kits that include oligonucleotide primers foran in vitro amplification reaction, the illustrated embodiments provideguidelines for the amounts useful for a single amplification reaction.In addition, those of ordinary skill in the art know the amount ofoligonucleotide primer that is appropriate for use in a singleamplification reaction. General guidelines may for instance be found inInnis et al. (PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc., San Diego, Calif., 1990), Sambrook et al. (InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989),and Ausubel et al. (In Current Protocols in Molecular Biology, GreenePubl. Assoc. and Wiley-Intersciences, 1992).

[0425] In some embodiments, kits may also include one or more of thereagents necessary to carry out PCR amplification reactions, including,for instance, DNA sample preparation reagents, appropriate buffers(e.g., polymerase buffer), salts (e.g., magnesium chloride), anddeoxyribonucleotides (dNTPs). In some embodiments, kits may include oneor more of the reagents necessary to carry out a transformationreaction. For instance, in kits that are tailored to be used with ayeast (particularly S. cerevisiae) system, the kit may include lithiumacetate or other buffers or reagents used in yeast transformation.

[0426] It may also be advantageous to provide in the kit one or morepositive or negative control nucleic acid molecules for use in in vitroamplification or transformation reactions. The design of such controlnucleic acid molecules is known to those of ordinary skill in the art.In some embodiments, a control strain may be provided, for instance anegative control strain that is not competent for recombination (e.g., ayeast rad52 strain). Similarly, an appropriate positive control strainmight be one that has incorporated into its genome a CORE-cassette thatpermits detection of successful (or failed) IRO transformation. By wayof example, one representative positive control strain is a yeast strain(such as BY4742) containing the CORE-cassette (or a CORE-DSB-cassette,or a RE-DSB-cassette, depending on the kit) in the existing TRP5 mutantgene so that transformation with IROs TRP5.e and TRP5.f (as describedabove) results in cells showing a Trp+ phenotype and carrying a silentmutation at TRP5 with a new a BamHI site.

[0427] This disclosure provides methods for site directed and/or randomin vivo mutagenesis, including point mutations, deletions, insertions,and replacements. The disclosure further provides components necessaryor useful for such mutagenesis methods, and kits for carrying out suchmethods. It will be apparent that the precise details of the methodsdescribed may be varied or modified without departing from the spirit ofthe described invention. We claim all such modifications and variationsthat fall within the scope and spirit of the claims below.

1 83 1 20 DNA Artificial sequence Synthetic oligonucleotide 1 gagctcgttttcgacactgg 20 2 20 DNA Artificial sequence Synthetic oligonucleotide 2tccttaccat taagttgatc 20 3 60 DNA Artificial sequence Syntheticoligonucleotide 3 aatttaaggc gaaagaacac tgggcggaag caaaccggca gagctcgttttcgacactgg 60 4 60 DNA Artificial sequence Synthetic oligonucleotide 4gatgtttcat atttatataa ttacattgtt taatattaca tccttaccat taagttgatc 60 562 DNA Artificial sequence Synthetic oligonucleotide 5 tgtttattatttttagtata caactatata gataatttac atgagctcgt tttcgacact 60 gg 62 6 60 DNAArtificial sequence Synthetic oligonucleotide 6 acagtttttc ttggctcctaaatttaatga cgaagaataa tccttaccat taagttgatc 60 7 70 DNA Artificialsequence Synthetic oligonucleotide 7 cttcatgcat gtctaagaga gttggaaaagggttttgatg aagctgtcgc gagctcgttt 60 tcgacactgg 70 8 70 DNA Artificialsequence Synthetic oligonucleotide 8 ggccaatata agaatacaag gatttgaagtcttcccagaa tgtgggatcg tccttaccat 60 taagttgatc 70 9 70 DNA Artificialsequence Synthetic oligonucleotide 9 gctagaaaaa ttttggtcct gactggtgcaggtgtttcaa cttcattagg gagctcgttt 60 tcgacactgg 70 10 70 DNA Artificialsequence Synthetic oligonucleotide 10 aatgtttgat ctttgaatag aacccctcagaagatctgaa gtccgggatc tccttaccat 60 taagttgatc 70 11 70 DNA Artificialsequence Synthetic oligonucleotide 11 tattgagaaa ttatactcaa aacattgataatttggaatc ttatgcggga gagctcgttt 60 tcgacactgg 70 12 70 DNA Artificialsequence Synthetic oligonucleotide 12 gtggcagtag caaaagagcc atggcactgcaccagtttat ctgtgcttat tccttaccat 60 taagttgatc 70 13 20 DNA Artificialsequence Synthetic oligonucleotide 13 agtcgtcact catggtgatt 20 14 20 DNAArtificial sequence Synthetic oligonucleotide 14 agacgacaaa ggcgatgcat20 15 20 DNA Artificial sequence Synthetic oligonucleotide 15 tcgttaatgaacagtggata 20 16 20 DNA Artificial sequence Synthetic oligonucleotide 16tcttctgctc agaagattca 20 17 20 DNA Artificial sequence Syntheticoligonucleotide 17 acactcaata ttcaacctga 20 18 20 DNA Artificialsequence Synthetic oligonucleotide 18 tcctttatct tcgatggcct 20 19 20 DNAArtificial sequence Synthetic oligonucleotide 19 tcgtggatat ggaacatcct20 20 20 DNA Artificial sequence Synthetic oligonucleotide 20 tggatgtggaaaccctgaat 20 21 20 DNA Artificial sequence Synthetic oligonucleotide 21ctgacggtgt agtgattggt 20 22 20 DNA Artificial sequence Syntheticoligonucleotide 22 catggcagca aactgttcct 20 23 20 DNA Artificialsequence Synthetic oligonucleotide 23 caactcgcaa gagcgtgttc 20 24 20 DNAArtificial sequence Synthetic oligonucleotide 24 tttgagaacg ccatatgagt20 25 20 DNA Artificial sequence Synthetic oligonucleotide 25 cggacttcagatcttctgag 20 26 20 DNA Artificial sequence Synthetic oligonucleotide 26gattccaaat tatcaatgtt 20 27 80 DNA Artificial sequence Syntheticoligonucleotide 27 ttctttttct agtggaagtt taccaaaaga aatttaaggcgaaagaacac tgggcggaag 60 caaaccggca tgtaatatta 80 28 80 DNA Artificialsequence Synthetic oligonucleotide 28 taatgacatt agtgacattt aaaatatgtagatgtttcat atttatataa ttacattgtt 60 taatattaca tgccggtttg 80 29 80 DNAArtificial sequence Synthetic oligonucleotide 29 ctttcgccgt cctttttcactcacagcaac aagcagcaag cactaagtac gcagtcaaaa 60 gagagaaaaa atgtaaatta 8030 80 DNA Artificial sequence Synthetic oligonucleotide 30 tttaaaacaaactttactgt tttttttttg tttattattt ttagtataca actatataga 60 taatttacattttttctctc 80 31 80 DNA Artificial sequence Synthetic oligonucleotide 31gtcagtatgt cccagaagct cttcatgcat gtctaagaga gttggaaaag ggttttgatg 60aagctgtcgc ggatcccaca 80 32 80 DNA Artificial sequence Syntheticoligonucleotide 32 tttgtgtagt gaagaaggac ggccaatata agaatacaaggatttgaagt cttcccagaa 60 tgtgggatcc gcgacagctt 80 33 80 DNA Artificialsequence Synthetic oligonucleotide 33 tgtgggatcc gcgacagctt catcaaaacccttttccaac tctcttagac atgcatgaag 60 agcttctggg acatactgac 80 34 80 DNAArtificial sequence Synthetic oligonucleotide 34 aagctgtcgc ggatcccacattctgggaag acttcaaatc cttgtattct tatattggcc 60 gtccttcttc actacacaaa 8035 95 DNA Artificial sequence Synthetic oligonucleotide 35 catgcatgtctaagagagtt ggaaaagggt tttgatgaag ctgtcgcgga tcccacattc 60 tgggaagacttcaaatcctt gtattcttat attgg 95 36 95 DNA Artificial sequence Syntheticoligonucleotide 36 ccaatataag aatacaagga tttgaagtct tcccagaatgtgggatccgc gacagcttca 60 tcaaaaccct tttccaactc tcttagacat gcatg 95 37 81DNA Artificial sequence Synthetic oligonucleotide 37 gtctaagagagttggaaaag ggttttgatg aagctgtcgc ggatcccaca ttctgggaag 60 acttcaaatccttgtattct t 81 38 81 DNA Artificial sequence Synthetic oligonucleotide38 aagaatacaa ggatttgaag tcttcccaga atgtgggatc cgcgacagct tcatcaaaac 60ccttttccaa ctctcttaga c 81 39 51 DNA Artificial sequence Syntheticoligonucleotide 39 aaaagggttt tgatgaagct gtcgcggatc ccacattctgggaagacttc a 51 40 51 DNA Artificial sequence Synthetic oligonucleotide40 tgaagtcttc ccagaatgtg ggatccgcga cagcttcatc aaaacccttt t 51 41 80 DNAArtificial sequence Synthetic oligonucleotide 41 tactggacga atttgatgaaaatcacaagc acccaattag atttggggac tttggtggtc 60 agtatgtccc agaagctctt 8042 80 DNA Artificial sequence Synthetic oligonucleotide 42 gatcttctctcttcaaccag atttgagcac cttgacaatg ctcagttaat ctctcagctt 60 tgtgtagtgaagaaggacgg 80 43 80 DNA Artificial sequence Synthetic oligonucleotide 43ttattcaaaa attacatacc gctagaaaaa ttttggtcct gactggtgca ggtgtttcaa 60cttcattagc gatcccggac 80 44 80 DNA Artificial sequence Syntheticoligonucleotide 44 ttggggatca tcgagcccca aatgtttgat ctttgaatagaacccctcag aagatctgaa 60 gtccgggatc gctaatgaag 80 45 95 DNA Artificialsequence Synthetic oligonucleotide 45 tagaaaaatt ttggtcctga ctggtgcaggtgtttcaact tcattagcga tcccggactt 60 cagatcttct gaggggttct attcaaagatcaaac 95 46 95 DNA Artificial sequence Synthetic oligonucleotide 46gtttgatctt tgaatagaac ccctcagaag atctgaagtc cgggatcgct aatgaagttg 60aaacacctgc accagtcagg accaaaattt ttcta 95 47 95 DNA Artificial sequenceSynthetic oligonucleotide 47 tagtttcatt aagatgctac aaatgaaagg gaaattattgagaaattata ctcaagccat 60 tgataatttg gaatcttatg cgggaataag cacag 95 48 80DNA Artificial sequence Synthetic oligonucleotide 48 tccaatggcaggtaacgcag gtggcagtag caaaagagcc atggcactgc accagtttat 60 ctgtgcttattcccgcataa 80 49 80 DNA Artificial sequence Synthetic oligonucleotide 49gctacaaatg aaagggaaat tattgagaaa ttatactcaa aacattgata atttggaatc 60ttatgcggga ataagcacag 80 50 95 DNA Artificial sequence Syntheticoligonucleotide 50 tctcaccggg taggttccaa tggcaggtaa cgcaggtggcagtagcaaaa gagccatagc 60 actgcaccag tttatctgtg cttattcccg cataa 95 51 22DNA Artificial sequence Synthetic oligonucleotide 51 tggaactcaatgaacctaag ga 22 52 20 DNA Artificial sequence Synthetic oligonucleotide52 agtgtggcaa cgccagcagt 20 53 20 DNA Artificial sequence Syntheticoligonucleotide 53 cttgaccaac gtggtcacct 20 54 20 DNA Artificialsequence Synthetic oligonucleotide 54 gtaaggccat tgaagatgca 20 55 60 DNAArtificial sequence Synthetic oligonucleotide 55 catagataca caggtcaataacctacaatg gttaatatct tcttcacagg cctcatgcgt 60 56 60 DNA Artificialsequence Synthetic oligonucleotide 56 aaaagcaacc gtcgatggtg accatttagcagaggcgaag acgcatgagg cctgtgaaga 60 57 60 DNA Artificial sequenceSynthetic oligonucleotide 57 agaatacaag gatttgaagt cttcccagaa tgtgggatcgtcttcacagg cctcatgcgt 60 58 60 DNA Artificial sequence Syntheticoligonucleotide 58 gtctaagaga gttggaaaag ggttttgatg aagctgtcgcacgcatgagg cctgtgaaga 60 59 20 DNA Artificial sequence Syntheticoligonucleotide 59 gggaaggtat ggaaggcaga 20 60 20 DNA Artificialsequence Synthetic oligonucleotide 60 tagtcgtcat caaaccagga 20 61 70 DNAArtificial sequence Synthetic oligonucleotide 61 cttcatgcat gtctaagagagttggaaaag ggttttgatg aagctgtcgc ttcgtacgct 60 gcaggtcgac 70 62 70 DNAArtificial sequence Synthetic oligonucleotide 62 ggccaatata agaatacaaggatttgaagt cttcccagaa tgtgggatcg ccgcgcgttg 60 gccgattcat 70 63 34 DNAArtificial sequence Synthetic oligonucleotide 63 gtcggaagat ctcgttggatggacgcaaag aagt 34 64 38 DNA Artificial sequence Syntheticoligonucleotide 64 tctgtgagat ctgacttatt atttcaggaa agtttcgg 38 65 80DNA Artificial sequence Synthetic oligonucleotide 65 taagaatacaaggatttgaa gtcttcccag aatgtgggat cgtagggata acagggtaat 60 ttggatggacgcaaagaagt 80 66 88 DNA Artificial sequence Synthetic oligonucleotide 66cttcatgcat gtctaagaga gttggaaaag ggttttgatg aagctgtcgc attaccctgt 60tatccctatt cgtacgctgc aggtcgac 88 67 88 DNA Artificial sequenceSynthetic oligonucleotide 67 cttcatgcat gtctaagaga gttggaaaag ggttttgatgaagctgtcgc tagggataac 60 agggtaattt cgtacgctgc aggtcgac 88 68 100 DNAArtificial sequence Synthetic oligonucleotide 68 taagaataca aggatttgaagtcttcccag aatgtgggat cgtagggata acagggtaat 60 ggattaccct gttatccctattggatggac gcaaagaagt 100 69 654 DNA Artificial sequence Synthetic GALpromoter 69 ttggatggac gcaaagaagt ttaataatca tattacatgg cattaccaccatatacatat 60 ccatatacat atccatatct aatcttactt atatgttgtg gaaatgtaaagagccccatt 120 atcttagcct aaaaaaacct tctctttgga actttcagta atacgcttaactgctcattg 180 ctatattgaa gtacggatta gaagccgccg agcgggtgac agccctccgaaggaagactc 240 tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa acgcagatgtgcctcgcgcc 300 gcactgctcc gaacaataaa gattctacaa tactagcttt tatggttatgaagaggaaaa 360 attggcagta acctggcccc acaaaccttc aaatgaacga atcaaattaacaaccatagg 420 atgataatgc gattagtttt ttagccttat ttctggggta attaatcagcgaagcgatga 480 tttttgatct attaacagat atataaatgc aaaaactgca taaccactttaactaatact 540 ttcaacattt tcggtttgta ttacttctta ttcaaatgta ataaaagtatcaacaaaaaa 600 ttgttaatat acctctatac tttaacgtca aggagaaaaa accccggatccatg 654 70 70 DNA Artificial sequence Synthetic oligonucleotide 70cttcatgcat gtctaagaga gttggaaaag ggttttgatg aagctgtcgc catctgggca 60gatgatgtcg 70 71 88 DNA Artificial sequence Synthetic oligonucleotide 71cttcatgcat gtctaagaga gttggaaaag ggttttgatg aagctgtcgc attaccctgt 60tatccctaca tctgggcaga tgatgtcg 88 72 88 DNA Artificial sequenceSynthetic oligonucleotide 72 cttcatgcat gtctaagaga gttggaaaag ggttttgatgaagctgtcgc tagggataac 60 agggtaatca tctgggcaga tgatgtcg 88 73 100 DNAArtificial sequence Synthetic oligonucleotide 73 taagaataca aggatttgaagtcttcccag aatgtgggat cgtagggata acagggtaat 60 ggattaccct gttatccctattggatggac gcaaagaagt 100 74 22 DNA Artificial sequence Syntheticoligonucleotide 74 tacaatcgat agattgtcgc ac 22 75 20 DNA Artificialsequence Synthetic oligonucleotide 75 ttcaatagct catcagtcga 20 76 23 DNAArtificial sequence Synthetic oligonucleotide 76 ccatggcctc cgcgaccggctgc 23 77 20 DNA Artificial sequence Synthetic oligonucleotide 77tgactgtacc accatccact 20 78 20 DNA Artificial sequence Syntheticoligonucleotide 78 ctgttcgatg ttcagttcga 20 79 24 DNA Artificialsequence Synthetic oligonucleotide 79 gcaggatcgc cgcggctccg ggcg 24 8041 DNA Artificial sequence Synthetic oligonucleotide 80 ggttttgatgaagctgtcgc ggatcccaca ttctgggaag a 41 81 41 DNA Artificial sequenceSynthetic oligonucleotide 81 tcttcccaga atgtgggatc cgcgacagct tcatcaaaacc 41 82 31 DNA Artificial sequence Synthetic oligonucleotide 82tgatgaagct gtcgcggatc ccacattctg g 31 83 31 DNA Artificial sequenceSynthetic oligonucleotide 83 ccagaatgtg ggatccgcga cagcttcatc a 31

1. A method for introducing a mutation into a target double strandednucleic acid sequence in a cell, wherein the double stranded nucleicacid sequence comprises a first and a second strand, the methodcomprising: introducing a double-stranded nucleic acid cassette into atarget nucleic acid sequence at an insertion point, wherein the cassetteis a RE-cassette and comprises a first portion homologous to a nucleicacid sequence on a first side of the insertion point; a second portionhomologous to a second nucleic acid sequence on a second side of theinsertion point; and a nucleic acid sequence encoding a reporter locatedbetween the first portion and the second portion; transforming the cellwith a first oligonucleotide comprising: a nucleic acid sequencehomologous to one strand (the chosen strand) of the target nucleic acidsequence at a position on the first side of the insertion point; and anucleic acid sequence homologous to the same strand of the targetnucleic acid sequence at a position on the second side of the insertionpoint, and comprising at least one nucleotide that differs from thechosen strand of the target nucleic acid sequence; and selecting forloss of the nucleic acid sequence encoding the reporter gene, whereinloss of the nucleic acid sequence encoding the reporter gene indicatesintegration of the oligonucleotide sequence comprising the at least onenucleotide that differs from the target nucleic acid sequence.
 2. Themethod of claim 1, wherein the double-stranded nucleic acid cassettefurther comprises a nucleic acid sequence encoding a counterselectablemarker located between the first portion and the second portion, whichcassette is referred to as a CORE-cassette, and wherein the methodfurther comprises selecting for loss of both the nucleic acid encodingthe counterselectable marker and the nucleic acid sequence encoding thereporter gene, wherein loss of both the nucleic acid sequence encodingthe counterselectable marker and the nucleic acid sequence encoding thereporter gene indicates integration of the oligonucleotide sequencecomprising the at least one nucleotide that differs from the targetnucleic acid sequence.
 3. The method of claim 1, wherein thedouble-stranded nucleic acid cassette further comprises a nucleic acidsequence comprising a double-strand break recognition site, a nucleicacid encoding a double-strand break enzyme that recognizes thedouble-strand break recognition site, and an inducible promoter,operably connected with the nucleic acid encoding the double-strandbreak enzyme, which cassette is referred to as a RE-DSB-cassette.
 4. Themethod of claim 2, wherein the double-stranded nucleic acid cassettefurther comprises a nucleic acid sequence comprising a double-strandbreak recognition site, a nucleic acid encoding a double-strand breakenzyme that recognizes the double-strand break recognition site, and aninducible promoter, operably connected with the nucleic acid encodingthe double-strand break enzyme, which cassette is referred to as aCORE-DSB-cassette.
 5. The method of claim 3 or 4, which method furthercomprises: inducing expression of the double strand break enzyme,thereby stimulating a double-strand break within the cassette, whichdouble-strand break stimulates recombination.
 6. The method of any oneof claims 1 through 4, wherein the oligonucleotide sequence comprisesmore than one nucleotide that differs from the target nucleic acidsequence.
 7. The method of claim 2 or 4, wherein transforming the cellwith the first oligonucleotide occurs prior to selecting for loss ofboth the nucleic acid encoding the counterselectable marker and thenucleic acid encoding the reporter gene.
 8. The method of any one ofclaims 1 through 4, further comprising transforming the cell with asecond oligonucleotide that is at least partially complementary to thefirst oligonucleotide.
 9. The method of claim 8, wherein transformingthe cell with the first oligonucleotide occurs concurrently withtransforming the cell with the second oligonucleotide.
 10. The method ofclaim 8, wherein the second oligonucleotide comprises: a nucleic acidsequence homologous to the target nucleic acid sequence at a position tothe first side of the insertion point.
 11. The method of claim 8,wherein the second oligonucleotide comprises: a nucleic acid sequencehomologous to the target nucleic acid sequence at a position to thesecond side of the insertion point.
 12. The method of claim 8, whereinthe second oligonucleotide comprises: a nucleic acid sequence homologousto the target nucleic acid sequence at a position to the first side ofthe insertion point; and a nucleic acid sequence homologous to thetarget nucleic acid sequence at a position to the second side of theinsertion point.
 13. The method of claim 8, wherein the first and/orsecond oligonucleotide contains at least one random nucleotide changecompared to the target nucleic acid sequence.
 14. The method of claim 8,wherein the second oligonucleotide is fully complementary to the firstoligonucleotide.
 15. The method of claim 8, where the 3′ ends of the twooligonucleotides are complementary but the first oligonucleotide lackshomology to the second side of the insertion point and the secondoligonucleotide lacks homology to the first side of the insertion point.16. The method of claim 8, where the 3′ ends of the two oligonucleotidesare complementary but the second oligonucleotide lacks homology to thesecond side of the insertion point and the first oligonucleotide lackshomology to the first side of the insertion point.
 17. The method ofclaim 8, where the 5′ ends of the two oligonucleotides are complementarybut the first oligonucleotide lacks homology to the second side of theinsertion point and the second oligonucleotide lacks homology to thefirst side of the insertion point.
 18. The method of claim 8, where the5′ ends of the two oligonucleotides are complementary but the secondoligonucleotide lacks homology to the second side of the insertion pointand the first oligonucleotide lacks homology to the first side of theinsertion point.
 19. The method of claim 2 or 4, wherein thecounterselectable marker is KlURA3, URA3, TRP5, TRP1, or a gene encodinga toxin.
 20. The method of claim 19, wherein the counterselectablemarker is a gene encoding a toxin, and the toxin is an induciblerestriction enzyme or an inducible p53 gene.
 21. The method of claim 20,wherein the inducible p53 gene is a toxic version.
 22. The method of anyone of claims 1 through 4, wherein the reporter encodes a polypeptidethat confers antibiotic resistance to the cell.
 23. The method of claim22, wherein the antibiotic is G418, hygromycin, kanamycin, ampicillin,tetracycline, chloramphenicol, neomycin, zeocin, nourseothricin,cycloheximide or canavanine.
 24. The method of any one of claims 1through 4, wherein the reporter encodes a polypeptide from an amino acidor nucleotide synthesis pathway.
 25. The method of claim 24, wherein thepolypeptide is LEU2, TRP5, TRP1, LYS2, HIS3, or ADE2.
 26. The method ofany one of claims 1 through 4, wherein the oligonucleotide is at least30 nucleotides in length.
 27. The method of claim 26, wherein theoligonucleotide is at least 40 nucleotides in length.
 28. The method ofclaim 26, wherein the oligonucleotide is at least 50 nucleotides inlength.
 29. The method of claim 26, wherein the oligonucleotide is atleast 80 nucleotides in length.
 30. The method of claim 8 or 16, whereinthe first and the second oligonucleotides are each at least 30nucleotides in length.
 31. The method of claim 30, wherein the first andthe second oligonucleotides are each at least 40 nucleotides in length.32. The method of claim 30, wherein the first and the secondoligonucleotides are at least 50 nucleotides in length.
 33. The methodof claim 30, wherein the first and the second oligonucleotides are atleast 80 nucleotides in length.
 34. The method of claim 8 or 16, whereinthe first and the second oligonucleotide are of different lengths. 35.The method of claim 8 or 16, wherein the region of overlap at the 3′ends is at least 10 base pairs.
 36. The method of claim 35, wherein theregion of overlap at the 3′ ends is at least 15 base pairs.
 37. Themethod of claim 36, wherein the 3′ ends of the first and secondoligonucleotide can be extended by in vitro polymerization.
 38. Themethod of claim 8, wherein at least one oligonucleotide differs from thetarget nucleic acid sequence by more than one nucleotide.
 39. The methodof claim 16, wherein both oligonucleotides differ from the targetnucleic acid sequence by at least a single nucleotide.
 40. The method ofclaim 39, wherein at least one nucleotide difference is inside theregion of overlap between the first and second oligonucleotides.
 41. Themethod of claim 39, wherein at least one nucleotide difference isoutside the region of overlap between the first and secondoligonucleotides.
 42. A method of deleting a target double strandednucleic acid sequence from within a cell, wherein the target doublestranded nucleic acid sequence comprises a first and a second strand,the method comprising introducing a double-stranded nucleic acidcassette into a target nucleic acid sequence at an insertion point,wherein the cassette is a RE-cassette and comprises a nucleic acidsequence encoding a reporter located between a first portion and asecond portion of the RE-cassette; transforming the cell with a firstoligonucleotide comprising: a nucleic acid sequence homologous to thefirst strand of a nucleic acid 5′ of the nucleic acid of interest; and asequence homologous to the first strand of a nucleic acid 3′ of thenucleic acid sequence of interest; and selecting for loss of the nucleicacid sequence encoding the reporter gene, wherein loss of the nucleicacid sequence encoding the reporter gene from the target double strandednucleic acid indicates deletion of the target double stranded nucleicacid.
 43. The method of claim 42, wherein the double-stranded nucleicacid cassette further comprises a nucleic acid sequence encoding acounterselectable marker located between the first portion and thesecond portion, which cassette is referred to as a CORE-cassette, andwherein the method further comprises selecting for loss of both thenucleic acid sequence encoding the counterselectable marker and thenucleic acid sequence encoding the reporter gene, wherein loss of boththe nucleic acid sequence encoding the counterselectable marker and thenucleic acid sequence encoding the reporter gene indicates deletion ofthe target double stranded nucleic acid.
 44. The method of claim 42,wherein the double-stranded nucleic acid cassette further comprises anucleic acid sequence comprising a double-strand break recognition site,a nucleic acid encoding a double-strand break enzyme that recognizes thedouble-strand break recognition site, and an inducible promoter,operably connected with the nucleic acid encoding the double-strandbreak enzyme, which cassette is referred to as a RE-DSB-cassette. 45.The method of claim 43, wherein the double-stranded nucleic acidcassette further comprises a nucleic acid sequence comprising adouble-strand break recognition site, a nucleic acid encoding adouble-strand break enzyme that recognizes the double-strand breakrecognition site, and an inducible promoter, operably connected with thenucleic acid encoding the double-strand break enzyme, which cassette isreferred to as a CORE-DSB-cassette.
 46. The method of claim 44 or 45,which method further comprises: inducing expression of the double strandbreak enzyme, thereby stimulating a double-strand break within thecassette, which double-strand break stimulates recombination.
 47. Themethod of claim 42, wherein the target sequence that is deleted isbetween 1 and about 16,000 bp in length.
 48. The method of any one ofclaims 42 through 45, wherein: the first portion of the cassette ishomologous to a nucleic acid sequence on a first side of the insertionpoint; and the second portion of the cassette is homologous to a secondnucleic acid sequence on the second side of the insertion point.
 49. Themethod of any one of claims 42 through 45, further comprising:transforming the cell with a second oligonucleotide comprising: anucleic acid sequence homologous to the second strand of a nucleic acid5′ of the target double stranded nucleic acid sequence; wherein thesequence of the first oligonucleotide is homologous to at least 10nucleotides at the 3′ end of the sequence of the second oligonucleotide.50. The method of claim 49, wherein the sequence of the firstoligonucleotide is homologous to at least 15 nucleotides at the 3′ endof the sequence of the second oligonucleotide.
 51. The method of claim49, wherein the second oligonucleotide further comprises: a sequencehomologous to the second strand of a nucleic acid 3′ of the nucleic acidsequence of interest.
 52. The method of claim 49, wherein the secondoligonucleotide is fully complementary to the first oligonucleotide. 53.The method of any one of claims 42 through 45, wherein thecounterselectable marker is KlURA3, URA3, TRP5, TRP1, or a gene encodinga toxin.
 54. The method of claim 49, wherein the counterselectablemarker is KlURA3, URA3, TRP5, TRP1, or a gene encoding a toxin.
 55. Themethod of claim 53, wherein the counterselectable marker is a geneencoding a toxin, and the toxin is an inducible restriction enzyme or aninducible p53 gene.
 56. The method of claim 55, wherein the induciblep53 gene is a toxic version.
 57. The method of any one of claims 42through 45 wherein the reporter encodes a polypeptide that confersantibiotic resistance to the cell.
 58. The method of claim 57, whereinthe antibiotic is G418, hygromycin, kanamycin, ampicillin, tetracycline,chloramphenicol, neomycin, zeocin, nourseothricin, cycloheximide orcanavanine.
 59. The method of any one of claims 42 through 45, whereinthe reporter encodes a polypeptide from an amino acid or nucleotidesynthesis pathway.
 60. The method of claim 59, wherein the polypeptideis LEU2, TRP5, TRP1, LYS2, HIS3, or ADE2.
 61. The method any one ofclaims 42 through 45, wherein the oligonucleotide is at least 30nucleotides in length.
 62. The method of claim 61, wherein theoligonucleotide is at least 40 nucleotides in length.
 63. The method ofclaim 61, wherein the oligonucleotide is at least 50 nucleotides inlength.
 64. The method of claim 61, wherein the oligonucleotide is atleast 80 nucleotides in length.
 65. The method of claim 49, wherein thefirst and the second oligonucleotides are each at least 20 nucleotidesin length.
 66. The method of claim 65, wherein the first and the secondoligonucleotides are each at least 30 nucleotides in length.
 67. Themethod of claim 65, wherein the first and the second oligonucleotidesare each at least 40 nucleotides in length.
 68. The method of claim 65,wherein the first and the second oligonucleotides are at least 50nucleotides in length.
 69. The method of claim 65, wherein the first andthe second oligonucleotides are at least 80 nucleotides in length. 70.The method of claim 65, wherein the first and the second oligonucleotideare of different lengths.
 71. The method of claim 65, wherein the firstand the second oligonucleotides are at least 20 nucleotides in lengthand wherein the region of overlap at the 3′ ends is at least 10 basepairs.
 72. The method of claim 65, wherein the first and the secondoligonucleotides are at least 40 nucleotides in length and wherein theregion of overlap at the 3′ ends is at least 15 base pairs.
 73. Themethod of claim 72, wherein the 3′ ends of the first and secondoligonucleotide can be extended by in vitro polymerization.
 74. Themethod of claim 1 through 4 or 42 through 45, wherein the cell is a cellof an organisms in which homologous recombination can be accomplished.75. The method of any one of claims 1 through 4 or 42 through 45,wherein the cell is a fungus cell, a bacteria cell, a plant cell, or ananimal cell.
 76. The method of claim 75, wherein the cell is a funguscell, and the fungus cell is a yeast cell.
 77. The method of claim 75,wherein the cell is an animal cell, and the animal cell is a chickencell.
 78. The method of claim 77, wherein the chicken cell comprises ahuman chromosome or fragment thereof.
 79. A method for assessing amutation in a nucleic acid sequence to determine if the mutation affectsa function or expression pattern of the nucleic acid, which methodcomprises the method of any one of claims 1 through 4 or 42 through 45.80. A method for analyzing a series of a mutations in a nucleic acidsequence, comprising performing the method of claim 79 a plurality oftimes, wherein at least two different mutations are introduced in thenucleic acid sequence; and analyzing the function or expression patternof the nucleic acid, thereby analyzing a series of a mutations in thenucleic acid sequence.
 81. The method of claim 79 or 80, wherein thenucleic acid sequence is a mammalian sequence.
 82. The method of claim79 or 80, wherein the nucleic acid sequence encodes a polypeptide. 83.The method of claim 82, wherein the method further comprises assessing afunction of the polypeptide.
 84. The method of claim 79 or 80, whichmethod is carried out in a cell of a haploid yeast.
 85. The method ofclaim 79 or 80, which method is carried out in a cell of a diploidyeast.
 86. The method of any one of claims 1 through 4 or 42 through 45,for use as a diagnostic tool wherein a series of strains or cell linesare created, each with the cassette at a different position within agene, such that mutations can be introduced anywhere within a gene andthe biological consequences assessed.
 87. The method of any one ofclaims 1 through 4 or 42 through 45, which method is a method ofanalyzing defects in p53 wherein a p53 mutant protein is expressed inyeast in such a way that an impact of a defect in the p53 mutant proteincan be assessed.
 88. The method of claim 87, further comprisingassessing a defect in the p53 mutant protein.
 89. A CORE-cassetteconstruct, a RE-DSB-cassette construct, or a CORE-DSB-cassette constructfor use in delitto perfetto mutagenesis.
 90. An integrative recombinantoligonucleotide (IRO) for use in delitto perfetto mutagenesis.
 91. A kitfor carrying out in vivo mutagenesis or deletion of a nucleic acidsequence, comprising an amount of a CORE-cassette construct, aRE-DSB-cassette construct, or a CORE-DSB-cassette construct, and anamount of an integrative recombinant oligonucleotide.